Qiagen handbooks

RNeasy® Mini Handbook

RNeasy Mini KitFor purification of total RNA from animal cells,animal tissues, bacteria, and yeast, and for RNA cleanup

RNeasy Protect Mini KitFor immediate stabilization of RNA in harvestedanimal tissues and subsequent total RNA purification

RNeasy Plant Mini KitFor purification of total RNA from plants andfilamentous fungi

Fourth Edition June 2012

Sample & Assay Technologies

QIAGEN Sample and Assay TechnologiesQIAGEN is the leading provider of innovative sample and assay technologies, enablingthe isolation and detection of contents of any biological sample. Our advanced,high-quality products and services ensure success from sample to result.QIAGEN sets standards in:■ Purification of DNA, RNA, and proteins■ Nucleic acid and protein assays■ microRNA research and RNAi■ Automation of sample and assay technologiesOur mission is to enable you to achieve outstanding success and breakthroughs. Formore information, visit www.qiagen.com.

ContentsKit Contents 4Storage 5Intended Use 5Safety Information 6Quality Control 6Introduction 7

Principle and procedure 8Description of protocols 10

Equipment and Reagents to Be Supplied by User 13Important Notes 16

Determining the amount of starting material 16Handling and storing starting material 18Disrupting and homogenizing starting material 18Eliminating genomic DNA contamination 21

ProtocolsPurification of Total RNA from Animal Cells using Spin Technology 23Purification of Total RNA from Animal Cells using Vacuum/Spin Technology 29Stabilization of RNA in Harvested Animal Tissues 34Purification of Total RNA from Animal Tissues 37Purification of Total RNA from Yeast 43Purification of Total RNA from Plant Cells and Tissues andFilamentous Fungi 50RNA Cleanup 54

Troubleshooting Guide 56Appendix A: General Remarks on Handling RNA 61Appendix B: Storage, Quantification, and Determination of Quality of RNA 63Appendix C: Formaldehyde Agarose Gel Electrophoresis 65Appendix D: Optional On-Column DNase Digestion with the RNase-Free DNase Set 67Appendix E: DNase Digestion of RNA before RNA Cleanup 69Appendix F: Acetone Precipitation of Protein from Buffer RLT Lysates 70Appendix G: RT-PCR and Real-Time RT-PCR 71Ordering Information 72

RNeasy Mini Handbook 06/2012 3

Kit Contents

RNeasy Mini Kit (50) (250)Catalog no. 74104 74106Number of preps 50 250RNeasy Mini Spin Columns (pink) 50 250Collection Tubes (1.5 ml) 50 250Collection Tubes (2 ml)* 50 250Buffer RLT*† 45 ml 220 mlBuffer RW1† 45 ml 220 mlBuffer RPE‡ (concentrate) 11 ml 65 mlRNase-Free Water 10 ml 50 mlQuick-Start Protocol 1 1

RNeasy Protect Mini Kit (50) (250)Catalog no. 74124 74126Number of preps 50 250RNAlater® RNA Stabilization Reagent* 50 ml 250 mlRNeasy Mini Spin Columns (pink) 50 250Collection Tubes (1.5 ml) 50 250Collection Tubes (2 ml)* 50 250Buffer RLT*† 45 ml 220 mlBuffer RW1† 45 ml 220 mlBuffer RPE‡ (concentrate) 11 ml 65 mlRNase-Free Water 10 ml 50 mlQuick-Start Protocol 1 1

* Also available separately. See page 72 for ordering information.† Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6 for safety

information.‡ Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a

working solution.

4 RNeasy Mini Handbook 06/2012

RNeasy Plant Mini Kit (20) (50)Catalog no. 74903 74904Number of preps 20 50RNeasy Mini Spin Columns (pink) 20 50QIAshredder Spin Columns (lilac) 20 50Collection Tubes (1.5 ml) 20 50Collection Tubes (2 ml)* 20 50Buffer RLT*† 18 ml 45 mlBuffer RLC† 18 ml 45 mlBuffer RW1† 18 ml 45 mlBuffer RPE‡ (concentrate) 5 ml 11 mlRNase-Free Water 10 ml 10 mlHandbook 1 1

* Also available separately. See page 72 for ordering information.† Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6 for safety

information.‡ Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a

working solution.

StorageThe RNeasy Mini Kit, RNeasy Protect Mini Kit (including RNAlater RNA StabilizationReagent), and RNeasy Plant Mini Kit should be stored dry at room temperature(15–25°C) and are stable for at least 9 months under these conditions.Storage of RNAlater Reagent at lower temperatures may cause precipitation. Beforeuse, redissolve the precipitate by heating to 37°C with agitation.

Intended UseThe RNeasy Mini Kit is intended for molecular biology applications. This product is notintended for the diagnosis, prevention, or treatment of a disease.All due care and attention should be exercised in the handling of the products. Werecommend all users of QIAGEN® products to adhere to the NIH guidelines that havebeen developed for recombinant DNA experiments, or to other applicable guidelines.



Safety InformationWhen working with chemicals, always wear a suitable lab coat, disposable gloves,and protective goggles. For more information, please consult the appropriate safety datasheets (SDSs). These are available online in convenient and compact PDF format atwww.qiagen.com/safety where you can find, view, and print the SDS for each QIAGENkit and kit component.

CAUTION: DO NOT add bleach or acidic solutions directly to thesample preparation waste.

Buffer RLT contains guanidine thiocyanate, Buffer RLC contains guanidinehydrochloride, and Buffer RW1 contains a small amount of guanidine thiocyanate.Guanidine salts can form highly reactive compounds when combined with bleach. Ifliquid containing these buffers is split, clean with suitable laboratory detergent andwater. If the spilt liquid contains potentially infectious agents, clean the affected areafirst with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.24-hour emergency informationEmergency medical information in English, French, and German can be obtained24 hours a day from:Poison Information Center Mainz, GermanyTel: +49-6131-19240

Quality ControlIn accordance with QIAGEN’s ISO-certified Quality Management System, each lot ofthe RNeasy Mini Kit, RNeasy Protect Mini Kit, and RNeasy Plant Mini Kit is testedagainst predetermined specifications to ensure consistent product quality.


IntroductionThe RNeasy Mini Handbook provides protocols for use with the following kits:

RNeasy Mini Kit — for purification of total RNA from animal cells, animal tissues,and yeast, and for cleanup of RNA from crude RNA preps and enzymaticreactions (e.g., DNase digestion, proteinase digestion, RNA ligation, and labelingreaction)RNeasy Protect Mini Kit — for immediate stabilization of RNA in harvested animaltissues and subsequent purification of total RNARNeasy Plant Mini Kit — for purification of total RNA from plant cells and tissuesand filamentous fungi

The RNeasy Mini Kit can also be used to purify total RNA from bacteria. In this case,we strongly recommend using the kit in combination with RNAprotect® Bacteria Reagent(available separately), which provides in vivo stabilization of RNA in bacteria to ensurereliable gene expression analysis. Various protocols for stabilizing and purifying RNAfrom different bacteria species are included in the RNAprotect Bacteria ReagentHandbook. The RNeasy Mini Kit and RNAprotect Bacteria Reagent can also bepurchased together as the RNeasy Protect Bacteria Mini Kit. For orderinginformation, see pages 74–75. It is also possible to use the RNeasy Mini Kit to purifycytoplasmic RNA from animal cells. The protocol can be downloaded atwww.qiagen.com/literature/protocols/RNeasyMini.aspx.The RNeasy Kits are designed to purify RNA from small amounts of starting material.They provide a fast and simple method for preparing up to 100 µg total RNA persample. The purified RNA is ready for use in downstream applications such as:

RT-PCR and real-time RT-PCRDifferential displaycDNA synthesisNorthern, dot, and slot blot analysesPrimer extensionPoly A+ RNA selectionRNase/S1 nuclease protectionMicroarraysRNA-Seq

The RNeasy Kits allow the parallel processing of multiple samples in less than30 minutes. Time-consuming and tedious methods, such as CsCl step-gradientultracentrifugation and alcohol precipitation, or methods involving the use of toxicsubstances, such as phenol and/or chloroform, are replaced by the RNeasy procedure.

Principle and procedureRNA purification using RNeasy technologyThe RNeasy procedure represents a well-established technology for RNA purification.This technology combines the selective binding properties of a silica-based membranewith the speed of microspin technology. A specialized high-salt buffer system allows upto 100 µg of RNA longer than 200 bases to bind to the RNeasy silica membrane.Biological samples are first lysed and homogenized in the presence of a highlydenaturing guanidine-thiocyanate–containing buffer, which immediately inactivatesRNases to ensure purification of intact RNA. Ethanol is added to provide appropriatebinding conditions, and the sample is then applied to an RNeasy Mini spin column,where the total RNA binds to the membrane and contaminants are efficiently washedaway. High-quality RNA is then eluted in 30–100 µl water.With the RNeasy procedure, all RNA molecules longer than 200 nucleotides arepurified. The procedure provides an enrichment for mRNA since most RNAs <200nucleotides (such as 5.8S rRNA, 5S rRNA, and tRNAs, which together comprise15–20% of total RNA) are selectively excluded. The size distribution of the purified RNAis comparable to that obtained by centrifugation through a CsCl cushion, where smallRNAs do not sediment efficiently. Protocols for purification of small RNA using RNeasyKits are available at www.qiagen.com/goto/microRNAprotocols.In this handbook, different protocols are provided for different starting materials. Theprotocols differ primarily in the lysis and homogenization of the sample and in theadjustment of the conditions for binding RNA to the RNeasy membrane. Once thesample is bound to the membrane, the protocols are similar (see flowchart, next page).

RNA stabilization using RNAlater technologyRNA stabilization is an absolute prerequisite for reliable gene expression analysis.Immediate stabilization of RNA in biological samples is necessary because, directlyafter harvesting the samples, changes in the gene expression pattern occur due tospecific and nonspecific RNA degradation as well as to transcriptional induction. Suchchanges need to be avoided for all reliable quantitative gene expression analyses, suchas microarray analyses, quantitative RT-PCR, such as TaqMan® and LightCycler®

technology, and other nucleic acid-based technologies.


RNeasy MiniProcedure

RNeasy ProtectMini Procedure

RNeasy PlantMini Procedure


The RNeasy Protect Mini Kit is supplied with RNAlater RNA Stabilization Reagent,which represents a novel technology for the immediate preservation of the geneexpression pattern in animal tissues, enabling reliable gene expression analysis. Afterharvesting, tissues are immediately submerged in RNAlater RNA Stabilization Reagent,which rapidly permeates the tissues to stabilize and protect cellular RNA in situ. Thereagent preserves RNA for up to 1 day at 37°C, 7 days at room temperature(15–25°C), or 4 weeks at 2–8°C, allowing transportation, storage, and shipping ofsamples without ice or dry ice. Alternatively, the samples can be archived at –20°C or–80°C. During storage or transport in RNAlater RNA Stabilization Reagent, even atelevated temperatures (e.g., room temperature or 37°C), the cellular RNA remainsintact and undegraded. RNAlater technology allows large numbers of samples to beeasily processed and replaces inconvenient, dangerous, and equipment-intensivemethods, such as snap-freezing of samples in liquid nitrogen, storage at –80°C, cuttingand weighing on dry ice, or immediate processing of harvested samples.Note: RNAlater RNA Stabilization Reagent is not for stabilization of RNA in animalcells, whole blood, plasma, or serum.

Description of protocolsPurification of Total RNA from Animal Cells Using Spin TechnologyUp to 1 x 107 cells, depending on the cell line, are disrupted in Buffer RLT andhomogenized. An overview of disruption and homogenization methods is given onpages 18–21. Ethanol is then added to the lysate, creating conditions that promoteselective binding of RNA to the RNeasy membrane. The sample is then applied to theRNeasy Mini spin column. Total RNA binds to the membrane, contaminants areefficiently washed away, and high-quality RNA is eluted in RNase-free water. All bind,wash, and elution steps are performed by centrifugation in a microcentrifuge.

Purification of Total RNA from Animal Cells Using Vacuum/Spin TechnologyUp to 1 x 106 cells, depending on the cell line, are disrupted in Buffer RLT andhomogenized. An overview of disruption and homogenization methods is given onpages 18–21. Ethanol is then added to the lysate, creating conditions that promoteselective binding of RNA to the RNeasy membrane. The sample is then applied to theRNeasy Mini spin column. Total RNA binds to the membrane, contaminants areefficiently washed away, and high-quality RNA is eluted in RNase-free water. The bindand wash steps are performed on a QIAvac 24 or QIAvac 24 Plus manifold, and thefinal elution step is performed by centrifugation in a microcentrifuge.


Stabilization of RNA in Harvested Animal TissuesThis protocol describes how to stabilize RNA in harvested animal tissues using RNAlaterRNA Stabilization Reagent. Purification of total RNA from the stabilized tissues can besubsequently carried out according to “Protocol: Purification of Total RNA from AnimalTissues” (page 37).

Purification of Total RNA from Animal TissuesFresh, frozen, or RNAlater stabilized tissue (up to 30 mg, depending on the tissue type)is disrupted in Buffer RLT and homogenized. An overview of disruption andhomogenization methods is given on pages 18–21. Ethanol is then added to the lysate,creating conditions that promote selective binding of RNA to the RNeasy membrane.The sample is then applied to the RNeasy Mini spin column. Total RNA binds to themembrane, contaminants are efficiently washed away, and high-quality RNA is elutedin RNase-free water.

Purification of Total RNA from YeastThis protocol is for the purification of total RNA from up to 5 x 107 yeast cells. Twoalternative methods of disrupting yeast cell walls are provided: enzymatic lysis ormechanical disruption. In general, both methods function equally well. For someapplications, enzymatic lysis might be preferable since no additional laboratoryequipment is required. Mechanical disruption, however, is well-suited for time-courseexperiments where enzymatic digestion incubations are not practical.The enzymatic lysis method uses zymolase or lyticase digestion of the cell walls toconvert cells to spheroplasts, which are then used in the RNeasy procedure. For samplesof up to 5 x 107 yeast cells, spheroplasts are separated from the digestion mixture bycentrifugation before being lysed. For samples of up to 2 x 107 yeast cells, the digestionmixture is used directly in the RNeasy procedure without prior separation of thespheroplasts. After addition of Buffer RLT and ethanol, samples are loaded onto theRNeasy Mini spin column. Total RNA binds to the RNeasy membrane, contaminants areefficiently washed away, and high-quality RNA is eluted in RNase-free water.Using the mechanical disruption method, yeast cells are lysed and homogenized byhigh-speed agitation in the TissueLyser LT, TissueLyser II, or other bead mill in the presenceof glass beads and Buffer RLT. Ethanol is added to the lysate, creating conditions thatpromote selective binding of RNA to the RNeasy membrane. The sample is then appliedto the RNeasy Mini spin column. Total RNA binds to the membrane, contaminants areefficiently washed away, and high-quality RNA is eluted in RNase-free water.


Purification of Total RNA from Plant Cells and Tissues and Filamentous FungiUp to 100 mg of sample is first ground in liquid nitrogen and then lysed under highlydenaturing conditions. The RNeasy Plant Mini Kit provides a choice of lysis buffers:Buffer RLT and Buffer RLC, which contain guanidine thiocyanate and guanidinehydrochloride, respectively. The higher cell disruption and denaturing properties ofBuffer RLT frequently make it the buffer of choice. However, some tissues, such as milkyendosperm of maize or mycelia of filamentous fungi, solidify in Buffer RLT, making theextraction of RNA impossible. In these cases, Buffer RLC should be used instead. Afterlysis with either buffer, samples are centrifuged through a QIAshredder homogenizer.This simultaneously removes insoluble material and reduces the viscosity of the lysatesby disrupting gelatinous material often formed in plant and fungal lysates. Ethanol isadded to the cleared lysate, creating conditions which promote selective binding ofRNA to the RNeasy membrane. The sample is then applied to the RNeasy Mini spincolumn. Total RNA binds to the membrane, contaminants are efficiently washed away,and high-quality RNA is eluted in RNase-free water.

RNA CleanupThis protocol can be used to purify RNA from enzymatic reactions (e.g., DNasedigestion, RNA labeling) or to desalt RNA samples (up to 100 µg RNA). Buffer RLT andethanol are added to the sample to create conditions that promote selective binding ofRNA to the RNeasy membrane. The sample is then applied to the RNeasy Mini spincolumn. Total RNA binds to the membrane, contaminants are efficiently washed away,and high-quality RNA is eluted in RNase-free water.

Automated purificationPurification of RNA can be fully automated on the QIAcube®. The innovative QIAcube usesadvanced technology to process QIAGEN spin columns, enabling seamless integrationof automated, low-throughput sample prep into your laboratory workflow. Samplepreparation using the QIAcube follows the same steps as the manual procedure (i.e.,lyse, bind, wash, and elute), enabling you to continue using the RNeasy Mini Kit forpurification of high-quality RNA. For more information about the automated procedure,see the relevant protocol sheet available at www.qiagen.com/MyQIAcube.The QIAcube is preinstalled with protocols for purification of plasmid DNA, genomicDNA, RNA, viral nucleic acids, and proteins, plus DNA and RNA cleanup. The rangeof protocols available is continually expanding, and additional QIAGEN protocols canbe downloaded free of charge at www.qiagen.com/MyQIAcube.


Equipment and Reagents to Be Supplied by UserWhen working with chemicals, always wear a suitable lab coat, disposable gloves,and protective goggles. For more information, consult the appropriate safety data sheets(SDSs), available from the product supplier.

For all protocols14.3 M β-mercaptoethanol (β-ME) (commercially available solutions are usually14.3 M)Sterile, RNase-free pipet tipsMicrocentrifuge (with rotor for 2 ml tubes)96–100% ethanol*Disposable glovesEquipment for sample disruption and homogenization (see pages 18–21).Depending on the method chosen, one or more of the following are required:

Trypsin and PBSQIAshredder homogenizer (see ordering information, page 73Blunt needle and syringeMortar and pestleTissueLyser LT or TissueLyser II (see ordering information, page 73)Rotor–stator homogenizer

For RNA purification from animal cells70% ethanol*

For RNA purification from animal cells using vacuum technologyQIAvac 24 (no longer available); QIAvac 24 Plus (cat. no. 19413); or other vacuummanifold with luer connectors and capable of dealing with vacuum pressures of–800 to –900 mbarQIAGEN Vacuum Pump (see page 73 for ordering information); or other vacuumpump capable of generating a vacuum pressure of –800 to –900 mbar and witha capacity of 18–20 liter/minNote: Use of insufficient vacuum pressure may reduce RNA yield and purity. TheRNeasy procedure requires higher vacuum pressures compared with otherQIAGEN procedures. Most water pumps or house vacuums do not providesufficient vacuum pressure.

* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.


Optional: Vacuum Regulator (cat. no. 19530) to measure the pressure differencebetween the inside and outside of a vacuum systemA vacuum pressure of –800 to –900 mbar should develop when RNeasy Mini spincolumns are used on the vacuum manifold. Vacuum pressures exceeding–900 mbar should be avoided. The vacuum pressure is the pressure differencebetween the inside of the manifold and the atmosphere (standard atmosphericpressure: 1013 mbar or 760 mm Hg) and can be regulated and measured usinga pressure gauge or vacuum regulator. Vacuum recommendations are given innegative units to indicate the required reduction in pressure with respect to theatmosphere.Optional: VacConnectors (cat. no. 19407)These disposable connectors fit between the RNeasy Mini spin columns and theluer extensions on the QIAvac 24 or QIAvac 24 Plus. They prevent direct contactbetween the RNeasy Mini spin columns and luer connectors during RNA purification,avoiding any cross-contamination between samples. VacConnectors are discardedafter single use.

For RNA purification from animal tissues70% ethanol*Optional: Dithiothreitol (DTT)

For RNA purification from yeast using enzymatic lysis70% ethanol*Buffer for enzymatic lysisIn most cases, Buffer Y1 (containing sorbitol, EDTA, β-ME, and lyticase orzymolase) can be used. See the protocol on page 45 for details on preparingBuffer Y1.

For RNA purification from yeast using mechanical disruption70% ethanol*Glass beads, 0.45–0.55 mm diameterConcentrated nitric acid, deionized water, and baking ovenTissueLyser LT, TissueLyser II, or other bead-mill homogenizer

* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.


For RNA purification from plants and fungiLiquid nitrogenMortar and pestle (alternatively, TissueLyser LT, TissueLyser II, or other bead-millhomogenizer)

Suppliers of equipment for disruption and homogenization*Rotor–stator homogenizers can be purchased from:

BioSpec Products, Inc. (www.biospec.com): Tissue-Tearor™ homogenizerCharles Ross & Son Company (www.mixers.com)IKA (www.ika.de): ULTRA-TURRAX® dispersersKINEMATICA AG (www.kinematica.ch) or Brinkmann Instruments, Inc.(www.brinkmann.com): POLYTRON® laboratory dispersing devicesOmni International, Inc. (www.omni-inc.com)Silverson (www.silverson.com)VirTis (www.virtis.com)

Bead-mill homogenizers and stainless steel and tungsten carbide beads can be pur-chased from:

QIAGEN (TissueLyser system, see page 73 for ordering information)Glass, stainless steel, and tungsten carbide beads can be purchased from:

Retsch (www.retsch.de)

* This is not a complete list of suppliers and does not include many important vendors of biological supplies.


Important NotesDetermining the amount of starting materialIt is essential to use the correct amount of starting material to obtain optimal RNA yieldand purity. The maximum amount that can be used is determined by:

The type of sample and its RNA contentThe volume of Buffer RLT required for efficient lysisThe RNA binding capacity of the RNeasy spin column

When processing samples containing high amounts of RNA, less than the maximumamount of starting material shown in Table 1 should be used, so that the RNA bindingcapacity of the RNeasy spin column is not exceeded.When processing samples containing average or low amounts of RNA, the maximumamount of starting material shown in Table 1 can be used. However, even though theRNA binding capacity of the RNeasy spin column is not reached, the maximum amountof starting material must not be exceeded. Otherwise, lysis will be incomplete andcellular debris may interfere with the binding of RNA to the RNeasy spin columnmembrane, resulting in lower RNA yield and purity.More information on using the correct amount of starting material is given in eachprotocol. Table 2 shows expected RNA yields from various sources.

Table 1. RNeasy Mini spin column specifications

Maximum binding capacity 100 µg RNAMaximum loading volume 700 µlRNA size distribution RNA >200 nucleotidesMinimum elution volume 30 µlMaximum amount of starting material

Animal cells 1 x 107*Animal tissues 30 mg*Yeast 5 x 107*Plant tissues 100 mgFilamentous fungi 100 mg

* For larger sample sizes, RNeasy Kits and RNeasy Protect Kits are available in midi and maxi formats. Fordetails, visit www.qiagen.com/RNA.


Note: If the binding capacity of the RNeasy spin column is exceeded, RNA yields willnot be consistent and may be reduced. If lysis of the starting material is incomplete,RNA yields will be lower than expected, even if the binding capacity of the RNeasyspin column is not exceeded.

Table 2. Typical yields of total RNA with RNeasy Mini spin columns

Source Yield of total RNA* (µg)Cell cultures (1 x 106 cells)

NIH/3T3 10HeLa 15COS-7 35LMH 12Huh 15

Mouse/rat tissues (10 mg)Embryo (13 day) 25Kidney 20–30Liver 40–60Spleen 30–40Thymus 40–50Lung 10–20

Yeast (1 x 107 cells)S. cerevisiae 25

Plants (100 mg leaves)Arabidopsis 35Maize 25Tomato 65Tobacco 60

* Amounts can vary due to factors such as species, developmental stage, and growth conditions. Since theRNeasy procedure enriches for mRNA and other RNA species >200 nucleotides, the total RNA yield doesnot include 5S rRNA, tRNA, and other low-molecular-weight RNAs, which make up 15–20% of totalcellular RNA.


Handling and storing starting materialRNA in animal and plant tissues is not protected after harvesting until the sample istreated with RNAlater RNA Stabilization Reagent (animal tissues only), flash-frozen, ordisrupted and homogenized in the presence of RNase-inhibiting or denaturingreagents. Otherwise, unwanted changes in the gene expression profile will occur. It istherefore important that tissue samples are immediately frozen in liquid nitrogen andstored at –70°C, or immediately immersed in RNAlater RNA Stabilization Reagent.Alternatively, use Allprotect Tissue Reagent, which provides immediate stabilization ofDNA, RNA, and protein in tissues samples at room temperature.The procedures for tissue harvesting and RNA protection should be carried out asquickly as possible. Frozen tissue samples should not be allowed to thaw duringhandling or weighing. After disruption and homogenization in Buffer RLT (lysis buffer),samples can be stored at –70°C for months.Animal and yeast cells can be pelleted and then stored at –70°C until required for RNApurification. However, if performing RNA purification from yeast cells with enzymaticlysis, only freshly harvested samples can be used.

Disrupting and homogenizing starting materialEfficient disruption and homogenization of the starting material is an absoluterequirement for all total RNA purification procedures. Disruption and homogenizationare 2 distinct steps:

Disruption: Complete disruption of cell walls and plasma membranes of cells andorganelles is absolutely required to release all the RNA contained in the sample.Different samples require different methods to achieve complete disruption.Incomplete disruption results in significantly reduced RNA yields.Homogenization: Homogenization is necessary to reduce the viscosity of thelysates produced by disruption. Homogenization shears high-molecular-weightgenomic DNA and other high-molecular-weight cellular components to create ahomogeneous lysate. Incomplete homogenization results in inefficient binding ofRNA to the RNeasy spin column membrane and therefore significantly reducedRNA yields.

Some disruption methods simultaneously homogenize the sample, while others requirean additional homogenization step. Table 3 (page 19) gives an overview of differentdisruption and homogenization methods, and is followed by a detailed description ofeach method. This information can be used as a guide to choose the appropriatemethods for your starting material.Note: After storage in RNAlater RNA Stabilization Reagent, tissues become slightlyharder than fresh or thawed tissues. Disruption and homogenization of these tissues,however, is usually not a problem.


Table 3. Disruption and homogenization methods

Disruption HomogenizationSample method method CommentsAnimal cells Addition of lysis TissueRuptor If processing ≤1 x 105

buffer or QIAshredder cells, lysate can behomogenizer* homogenized byor syringe and vortexingneedle

Animal TissueLyser LT or TissueLyser LT or The TissueLyser LT ortissues TissueLyser II TissueLyser II TissueLyser II gives results

comparable to using theTissueRuptor

TissueRuptor TissueRuptor Simultaneously disruptsand homogenizes

Mortar and pestle QIAshredder TissueRuptor usuallyhomogenizer* gives higher yields thanor syringe and mortar and pestleneedle

Yeast Enzymatic Vortexingdigestion of cellwall followed bylysis ofspheroplastsTissueLyser LT or TissueLyser LT or TissueLyser LT orTissueLyser II with TissueLyser II TissueLyser IIglass beads with glass beads simultaneously disrupts

and homogenizes; cannotbe replaced by vortexing

Plants and Mortar and pestle QIAshredder Mortar and pestlefilamentous homogenizer* cannot be replaced byfungi the TissueRuptor

* QIAshredder homogenizers are supplied in the RNeasy Plant Mini Kit and can be purchased separately foruse with the RNeasy Mini Kit or RNeasy Protect Mini Kit. See page 74 for ordering information.


Disruption and homogenization using the TissueLyser systemIn bead-milling, cells and tissues can be disrupted by rapid agitation in the presence ofbeads and lysis buffer. Disruption and simultaneous homogenization occur by theshearing and crushing action of the beads as they collide with the cells. Disruptionefficiency is influenced by:

Size and composition of beadsRatio of buffer to beadsAmount of starting materialSpeed and configuration of the TissueLyser LT or TissueLyser IIDisintegration time

For animal tissues, the optimal beads are 3–7 mm diameter stainless steel beads, andfor yeast cells, the optimal beads are 0.5 mm diameter glass beads. It is essential thatglass beads are prewashed in concentrated nitric acid. All other disruption parametersmust be determined empirically for each application. The protocol for RNA purificationfrom yeast (page 43) describes how to perform mechanical disruption of yeast cells withglass beads. For guidelines on disruption and homogenization of animal tissues usingthe TissueLyser system, refer to the TissueLyser LT Handbook or the TissueLyser Handbook.For other bead mills, please refer to suppliers’ guidelines for further details.Plant tissues can be disrupted using the TissueLyser LT or TissueLyser II, in combinationwith stainless steel or tungsten carbide beads. In this case, plant material, beads, anddisruption vessels must all be precooled in liquid nitrogen, and disruption is performedwithout lysis buffer.

Disruption and homogenization using the TissueRuptor or other rotor–stator homogenizersRotor–stator homogenizers thoroughly disrupt and simultaneously homogenize, in thepresence of lysis buffer, single samples of animal tissues in 15–90 seconds dependingon the toughness and size of the sample. Rotor–stator homogenizers can also be usedto homogenize cell lysates. The rotor turns at a very high speed, causing the sample tobe disrupted and homogenized by a combination of turbulence and mechanicalshearing. Foaming of the sample should be kept to a minimum by using properly sizedvessels, keeping the tip of the homogenizer submerged, and holding the immersed tipto the side of the tube. Rotor–stator homogenizers are available in different sizes andoperate with differently sized probes. Probes with diameters of 5 mm and 7 mm aresuitable for volumes up to 300 µl and can be used for homogenization inmicrocentrifuge tubes. Probes with a diameter of 10 mm or above require larger tubes.In addition, round-bottomed tubes allow more efficient homogenization than conical-bottomed tubes.


Disruption using a mortar and pestleFor disruption using a mortar and pestle, freeze the animal or plant tissue immediatelyin liquid nitrogen and grind to a fine powder under liquid nitrogen. Transfer thesuspension (tissue powder and liquid nitrogen) into a liquid-nitrogen–cooled,appropriately sized tube and allow the liquid nitrogen to evaporate without allowingthe sample to thaw. Add lysis buffer and continue as quickly as possible with thehomogenization according to one of the 2 methods below.Note: Grinding the sample using a mortar and pestle will disrupt the sample, but willnot homogenize it. Homogenization must be performed afterwards.

Homogenization using QIAshredder homogenizersUsing QIAshredder homogenizers is a fast and efficient way to homogenize cell andtissue lysates without cross-contamination of samples. Up to 700 µl of lysate is loadedonto a QIAshredder spin column placed in a 2 ml collection tube, and spun for2 minutes at maximum speed in a microcentrifuge. The lysate is homogenized as itpasses through the spin column. QIAshredder spin columns are supplied in the RNeasyPlant Mini Kit and can be purchased separately for use with the RNeasy Mini Kit andRNeasy Protect Mini Kit. See page 74 for ordering information.

Homogenization using a syringe and needleCell and tissue lysates can be homogenized using a syringe and needle. Lysate ispassed through a 20-gauge (0.9 mm) needle attached to a sterile plastic syringe at least5–10 times or until a homogeneous lysate is achieved. Increasing the volume of lysisbuffer may be required to facilitate handling and minimize loss.

Eliminating genomic DNA contaminationGenerally, DNase digestion is not required with RNeasy Kits since RNeasy silica-membrane technology efficiently removes most of the DNA without DNase treatment.However, further DNA removal may be necessary for certain RNA applications that aresensitive to very small amounts of DNA (e.g., TaqMan RT-PCR analysis with alow-abundance target). In these cases, residual DNA can be removed by optional on-column DNase digestion using the RNase-Free DNase Set (see Appendix D, page 67).The DNase is efficiently removed in subsequent wash steps. Alternatively, residual DNAcan be removed by a DNase digestion after RNA purification (see Appendix E,page 69). The DNase digestion can then be cleaned up, if desired, using “Protocol:RNA Cleanup” (page 54).The RNeasy Plus Mini Kit, which is designed for RNA purification from animal cells andtissues, integrates unique gDNA Eliminator spin columns with RNeasy technology.Genomic DNA is effectively removed in a single, rapid centrifugation step, avoidingthe need for DNase digestion. See page 74 for ordering information.



If the purified RNA will be used in real-time, two-step RT-PCR, we recommend using theQuantiTect® Reverse Transcription Kit. The kit provides a fast and convenient procedure,enabling cDNA synthesis and genomic DNA removal in only 20 minutes. For orderinginformation, see page 76.

AnimalCells

Spin

Protocol: Purification of Total RNA from Animal Cellsusing Spin TechnologyThis protocol requires the RNeasy Mini Kit.

Determining the correct amount of starting materialIt is essential to use the correct amount of starting material to obtain optimal RNA yieldand purity. The minimum amount is generally 100 cells, while the maximum amountdepends on:

The RNA content of the cell typeThe RNA binding capacity of the RNeasy spin column (100 µg RNA)The volume of Buffer RLT required for efficient lysis (the maximum volume of BufferRLT that can be used limits the maximum amount of starting material to 1 x 107

cells)RNA content can vary greatly between cell types. The following examples illustrate howto determine the maximum amount of starting material:

COS cells have high RNA content (approximately 35 µg RNA per 106 cells). Donot use more than 3 x 106 cells, otherwise the RNA binding capacity of the RNeasyspin column will be exceeded.HeLa cells have average RNA content (approximately 15 µg RNA per 106 cells).Do not use more than 7 x 106 cells, otherwise the RNA binding capacity of theRNeasy spin column will be exceeded.NIH/3T3 cells have low RNA content (approximately 10 µg RNA per 106 cells).The maximum amount of starting material (1 x 107 cells) can be used.

If processing a cell type not listed in Table 2 (page 17) and if there is no informationabout its RNA content, we recommend starting with no more than 3–4 x 106 cells.Depending on RNA yield and purity, it may be possible to increase the cell number insubsequent preparations.Do not overload the RNeasy spin column, as this will significantly reduce RNA yield andpurity.Counting cells is the most accurate way to quantitate the amount of starting material.As a guide, the number of HeLa cells obtained in various culture vessels after confluentgrowth is given in Table 4.


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Table 4. Growth area and number of HeLa Cells in various culture vessels

Cell-culture vessel Growth area (cm2)* Number of cells†

Multiwell-plates96-well 0.32–0.6 4–5 x 104

48-well 1 1 x 105

24-well 2 2.5 x 105

12-well 4 5 x 105

6-well 9.5 1 x 106

Dishes35 mm 8 1 x 106

60 mm 21 2.5 x 106

100 mm 56 7 x 106

145–150 mm 145 2 x 107

Flasks40–50 ml 25 3 x 106

250–300 ml 75 1 x 107

650–750 ml 162–175 2 x 107

* Per well, if multiwell plates are used; varies slightly depending on the supplier.† Cell numbers are given for HeLa cells (approximate length = 15 µm), assuming confluent growth. Cell

numbers will vary for different kinds of animal cells, which vary in length from 10 to 30 µm.

Important points before startingIf using the RNeasy Kit for the first time, read “Important Notes” (page 16).If working with RNA for the first time, read Appendix A (page 61).Cell pellets can be stored at –70°C for later use or used directly in the procedure.Determine the number of cells before freezing. Frozen cell pellets should bethawed slightly so that they can be dislodged by flicking the tube in step 2.Homogenized cell lysates from step 3 can be stored at –70°C for several months.Frozen lysates should be incubated at 37°C in a water bath until completelythawed and salts are dissolved. Avoid prolonged incubation, which maycompromise RNA integrity. If any insoluble material is visible, centrifuge for 5 minat 3000–5000 x g. Transfer supernatant to a new RNase-free glass orpolypropylene tube, and continue with step 4.

AnimalCells

SpinBuffer RLT may form a precipitate upon storage. If necessary, redissolve bywarming, and then place at room temperature (15–25°C).Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore notcompatible with disinfecting reagents containing bleach. See page 6 for safetyinformation.Perform all steps of the procedure at room temperature. During the procedure,work quickly.Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensurethat the centrifuge does not cool below 20°C.

Things to do before startingIf purifying RNA from cell lines rich in RNases, we recommend addingβ-mercaptoethanol (β-ME) to Buffer RLT before use. Add 10 µl β-ME per 1 ml BufferRLT. Dispense in a fume hood and wear appropriate protective clothing. Buffer RLTcontaining β-ME can be stored at room temperature (15–25°C) for up to 1 month.Buffer RPE is supplied as a concentrate. Before using for the first time, add4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a workingsolution.If performing optional on-column DNase digestion, prepare DNase I stock solutionas described in Appendix D (page 67).

Procedure1. Harvest cells according to step 1a or 1b.1a. Cells grown in suspension (do not use more than 1 x 107 cells):

Determine the number of cells. Pellet the appropriate number of cells bycentrifuging for 5 min at 300 x g in a centrifuge tube (not supplied). Carefullyremove all supernatant by aspiration, and proceed to step 2.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute thelysate, affecting the conditions for binding of RNA to the RNeasy membrane. Botheffects may reduce RNA yield.


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1b. Cells grown in a monolayer (do not use more than 1 x 107 cells):Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) ortrypsinized and collected as a cell pellet prior to lysis. Cells grown in cell-cultureflasks should always be trypsinized.To lyse cells directly:Determine the number of cells. Completely aspirate the cell-culture medium, andproceed immediately to step 2.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute thelysate, affecting the conditions for binding of RNA to the RNeasy membrane. Botheffects may reduce RNA yield.To trypsinize and collect cells:Determine the number of cells. Aspirate the medium, and wash the cells with PBS.Aspirate the PBS, and add 0.1–0.25% trypsin in PBS. After the cells detach fromthe dish or flask, add medium (containing serum to inactivate the trypsin), transferthe cells to an RNase-free glass or polypropylene centrifuge tube (not supplied),and centrifuge at 300 x g for 5 min. Completely aspirate the supernatant, andproceed to step 2.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute thelysate, affecting the conditions for binding of RNA to the RNeasy membrane. Botheffects may reduce RNA yield.

2. Disrupt the cells by adding Buffer RLT.For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add theappropriate volume of Buffer RLT (see Table 5). Vortex or pipet to mix, and proceedto step 3.Note: Incomplete loosening of the cell pellet may lead to inefficient lysis andreduced RNA yields.

Table 5. Volumes of Buffer RLT for lysing pelleted cells

Number of pelleted cells Volume of Buffer RLT (µl)<5 x 106 3505 x 106 – 1 x 107 600

For direct lysis of cells grown in a monolayer, add the appropriate volume of BufferRLT (see Table 6) to the cell-culture dish. Collect the lysate with a rubber policeman.Pipet the lysate into a microcentrifuge tube (not supplied). Vortex or pipet to mix,and ensure that no cell clumps are visible before proceeding to step 3.

AnimalCells

SpinTable 6. Volumes of Buffer RLT for direct cell lysis

Dish diameter (cm) Volume of Buffer RLT (µl)*<6 3506–10 600

* Regardless of the cell number, use the buffer volumes indicated to completely cover the surface of the dish.

3. Homogenize the lysate according to step 3a, 3b, or 3c.See “Disrupting and homogenizing starting material”, pages 18–21, for moredetails on homogenization. If processing ≤1 x 105 cells, homogenize by vortexingfor 1 min. After homogenization, proceed to step 4.Note: Incomplete homogenization leads to significantly reduced RNA yields andcan cause clogging of the RNeasy spin column. Homogenization with a rotor–stator or QIAshredder homogenizer generally results in higher RNA yields thanwith a syringe and needle.

3a. Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collectiontube, and centrifuge for 2 min at full speed. Proceed to step 4.

3b. Homogenize the lysate for 30 s using a rotor–stator homogenizer. Proceed tostep 4.

3c. Pass the lysate at least 5 times through a blunt 20-gauge needle (0.9 mm diameter)fitted to an RNase-free syringe. Proceed to step 4.

4. Add 1 volume of 70% ethanol to the homogenized lysate, and mix well bypipetting. Do not centrifuge.Note: The volume of lysate may be less than 350 µl or 600 µl due to loss duringhomogenization.Note: When purifying RNA from certain cell lines, precipitates may be visible afteraddition of ethanol. This does not affect the procedure.

5. Transfer up to 700 µl of the sample, including any precipitate that may haveformed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Closethe lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard theflow-through.*Reuse the collection tube in step 6.If the sample volume exceeds 700 µl, centrifuge successive aliquots in the sameRNeasy spin column. Discard the flow-through after each centrifugation.*Optional: If performing optional on-column DNase digestion (see “Eliminatinggenomic DNA contamination”, page 21), follow steps D1–D4 (page 67) afterperforming this step.

* Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6for safety information.


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6. Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, andcentrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin columnmembrane. Discard the flow-through.*Reuse the collection tube in step 7.Note: After centrifugation, carefully remove the RNeasy spin column from thecollection tube so that the column does not contact the flow-through. Be sure toempty the collection tube completely.Skip this step if performing optional on-column DNase digestion (page 67).

7. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, andcentrifuge for 15 s at≥8000 x g (≥10,000 rpm) to wash the spin column membrane.Discard the flow-through.Reuse the collection tube in step 8.Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added toBuffer RPE before use (see “Things to do before starting”).

8. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, andcentrifuge for 2 min at≥8000 x g (≥10,000 rpm) to wash the spin column membrane.The long centrifugation dries the spin column membrane, ensuring that no ethanolis carried over during RNA elution. Residual ethanol may interfere withdownstream reactions.Note: After centrifugation, carefully remove the RNeasy spin column from thecollection tube so that the column does not contact the flow-through. Otherwise,carryover of ethanol will occur.

9. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied),and discard the old collection tube with the flow-through. Close the lid gently, andcentrifuge at full speed for 1 min.Perform this step to eliminate any possible carryover of Buffer RPE, or if residualflow-through remains on the outside of the RNeasy spin column after step 8.

10. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add30–50 µl RNase-free water directly to the spin column membrane. Close the lidgently, and centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA.

11. If the expected RNA yield is >30 µg, repeat step 10 using another 30–50 µl RNase-free water, or using the eluate from step 10 (if high RNA concentration is required).Reuse the collection tube from step 10.If using the eluate from step 10, the RNA yield will be 15–30% less than thatobtained using a second volume of RNase-free water, but the final RNAconcentration will be higher.


AnimalCells

Vacuum/Spin

Protocol: Purification of Total RNA from Animal Cellsusing Vacuum/Spin TechnologyThis protocol requires the RNeasy Mini Kit.

Determining the correct amount of starting materialSee “Determining the correct amount of starting material”, page 16.

Important points before startingIf using the RNeasy Kit for the first time, read “Important Notes” (page 16).If working with RNA for the first time, read Appendix A (page 61).Cell pellets can be stored at –70°C for later use or used directly in the procedure.Determine the number of cells before freezing. Frozen cell pellets should bethawed slightly so that they can be dislodged by flicking the tube in step 2.Homogenized cell lysates from step 3 can be stored at –70°C for several months.Frozen lysates should be incubated at 37°C in a water bath until completelythawed and salts are dissolved. Avoid prolonged incubation, which maycompromise RNA integrity. If any insoluble material is visible, centrifuge for 5 minat 3000–5000 x g. Transfer supernatant to a new RNase-free glass orpolypropylene tube, and continue with step 4.Buffer RLT may form a precipitate upon storage. If necessary, redissolve bywarming, and then place at room temperature (15–25°C).Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore notcompatible with disinfecting reagents containing bleach. See page 6 for safetyinformation.Perform all steps of the procedure at room temperature. During the procedure,work quickly.Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensurethat the centrifuge does not cool below 20°C.Do not use more than 106 cells per prep. The cell numbers in each prep should besimilar (no more than a twofold difference between the highest and lowest) to allowuniform flow rates on the vacuum manifold.Between loading steps, switch off the vacuum and ventilate the manifold tomaintain uniform conditions for each sample. This can be done with a vacuumregulator inserted between the vacuum source and the vacuum manifold.Always use caution and wear safety glasses when working near a vacuummanifold under pressure.Always leave the lids of the RNeasy spin columns open while applying vacuum.


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The flow-through from each vacuum step is collected in the QIAvac 24 Plus or inthe QIAvac 24 base. Each can hold the waste from 24 samples. At the end of theprocedure, discard the liquid waste and clean the vacuum manifold as describedin the QIAvac 24 Plus Handbook or QIAvac Handbook. If using other vacuummanifolds, follow the supplier’s instructions.

Things to do before startingIf purifying RNA from cell lines rich in RNases, we recommend addingβ-mercaptoethanol (β-ME) to Buffer RLT before use. Add 10 µl β-ME per 1 ml BufferRLT. Dispense in a fume hood and wear appropriate protective clothing. Buffer RLTcontaining β-ME can be stored at room temperature (15–25°C) for up to 1 month.Buffer RPE is supplied as a concentrate. Before using for the first time, add4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a workingsolution.If performing optional on-column DNase digestion, prepare DNase I stock solutionas described in Appendix D (page 67).Set up the vacuum manifold according to the supplier’s instructions. If using theQIAvac Plus 24, refer to the QIAvac 24 Plus Handbook. If using the QIAvac 24,refer to the QIAvac Handbook. Insert each RNeasy spin column into a luerconnector.

Procedure1. Harvest cells according to step 1a or 1b.1a. Cells grown in suspension (do not use more than 1 x 106 cells):

Determine the number of cells. Pellet the appropriate number of cells bycentrifuging for 5 min at 300 x g in a centrifuge tube (not supplied). Carefullyremove all supernatant by aspiration, and proceed to step 2.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute thelysate, affecting the conditions for binding of RNA to the RNeasy membrane. Botheffects may reduce RNA yield.

AnimalCells

Vacuum/Spin

1b. Cells grown in a monolayer (do not use more than 1 x 106 cells):Cells can be either lysed directly in the cell-culture vessel (up to 10 cm diameter) ortrypsinized and collected as a cell pellet prior to lysis. Cells grown in cell-cultureflasks should always be trypsinized.To lyse cells directly:Determine the number of cells. Completely aspirate the cell-culture medium, andproceed immediately to step 2.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute thelysate, affecting the conditions for binding of RNA to the RNeasy membrane. Botheffects may reduce RNA yield.To trypsinize and collect cells:Determine the number of cells. Aspirate the medium, and wash the cells with PBS.Aspirate the PBS, and add 0.1–0.25% trypsin in PBS. After the cells detach fromthe dish or flask, add medium (containing serum to inactivate the trypsin), transferthe cells to an RNase-free glass or polypropylene centrifuge tube (not supplied),and centrifuge at 300 x g for 5 min. Completely aspirate the supernatant, andproceed to step 2.Note: Incomplete removal of cell-culture medium will inhibit lysis and dilute thelysate, affecting the conditions for binding of RNA to the RNeasy membrane. Botheffects may reduce RNA yield.

2. Disrupt the cells by adding Buffer RLT.For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Add 350 µlBuffer RLT. Vortex or pipet to mix, and proceed to step 3.Note: Incomplete loosening of the cell pellet may lead to inefficient lysis andreduced RNA yields.For direct lysis of cells grown in a monolayer, add 350 µl Buffer RLT to the cell-culture dish (if 350 µl is not enough to cover the dish, use 600 µl Buffer RLT instead;be sure then to use 600 µl of 70% ethanol in step 4). Collect the lysate with a rubberpoliceman. Pipet the lysate into a microcentrifuge tube (not supplied). Vortex orpipet to mix, and ensure that no cell clumps are visible before proceeding to step 3.

3. Homogenize the lysate according to step 3a, 3b, or 3c.See “Disrupting and homogenizing starting material”, pages 18–21, for moredetails on homogenization. If processing ≤1 x 105 cells, homogenize by vortexingfor 1 min. After homogenization, proceed to step 4.Note: Incomplete homogenization leads to significantly reduced RNA yields andcan cause clogging of the RNeasy spin column. Homogenization with a rotor–stator or QIAshredder homogenizer generally results in higher RNA yields thanwith a syringe and needle.


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3a. Pipet the lysate directly into a QIAshredder spin column placed in a 2 ml collectiontube, and centrifuge for 2 min at full speed. Proceed to step 4.

3b. Homogenize the lysate for 30 s using a rotor–stator homogenizer. Proceed tostep 4.

3c. Pass the lysate at least 5 times through a blunt 20-gauge needle (0.9 mm diameter)fitted to an RNase-free syringe. Proceed to step 4.

4. Add 1 volume of 70% ethanol to the homogenized lysate, and mix well bypipetting. Do not centrifuge.Note: The volume of lysate may be less than 350 µl or 600 µl due to loss duringhomogenization.Note: When purifying RNA from certain cell lines, precipitates may be visible afteraddition of ethanol. This does not affect the procedure.

5. Transfer 700 µl of each sample from step 4, including any precipitate that mayhave formed, to each RNeasy spin column on the vacuum manifold.

6. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off thevacuum and ventilate the vacuum manifold.Make sure that the vacuum manifold is assembled correctly before loading. Theflow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base.* If a spincolumn clogs, switch off the vacuum, ventilate, and try again. If it still clogs,continue with “Protocol: Purification of Total RNA from Animal Cells Using SpinTechnology”, page 23.Note: Be sure to switch off the vacuum and ventilate the manifold betweenpipetting steps to maintain uniform conditions for each sample.

7. If necessary, repeat steps 5 and 6 with the remaining volume (approx. 500 µl) ofeach sample.The flow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base.*

8. Add 700 µl Buffer RW1 to each RNeasy spin column.9. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off the

vacuum and ventilate the vacuum manifold.The flow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base.*

10. Add 500 µl Buffer RPE to each RNeasy spin column.Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added toBuffer RPE before use (see “Things to do before starting”).

11. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off thevacuum and ventilate the vacuum manifold.The flow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base.


AnimalCells

Vacuum/Spin

12. Add 500 µl Buffer RPE to each RNeasy spin column.13. Switch on the vacuum. Apply vacuum until transfer is complete. Switch off the

vacuum and ventilate the vacuum manifold.The flow-through is collected in the QIAvac 24 Plus or the QIAvac 24 base.

14. Remove the RNeasy spin columns from the vacuum manifold, and place each in a2 ml collection tube (supplied). Close the lids gently, and centrifuge at full speedfor 1 min.

15. Place each RNeasy spin column in a new 1.5 ml collection tube (supplied). Add30–50 µl RNase-free water directly to each spin column membrane. Close the lidsgently, and centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA.

16. If the expected RNA yield is >30 µg, repeat step 15 using another 30–50 µl RNase-free water, or using the eluate from step 15 (if high RNA concentration is required).Reuse the collection tubes from step 15.If using the eluate from step 15, the RNA yield will be 15–30% less than thatobtained using a second volume of RNase-free water, but the final RNAconcentration will be higher.


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Protocol: Stabilization of RNA in Harvested AnimalTissuesThis protocol describes how to stabilize and store human and animal tissues in RNAlaterRNA Stabilization Reagent, included in the RNeasy Protect Mini Kit. For RNApurification from stabilized tissues, see “Protocol: Purification of Total RNA from AnimalTissues”, page 37.

Important notes about RNAlater RNA Stabilization ReagentRNA in harvested animal tissue is not protected until the tissue is completely submergedin a sufficient volume of RNAlater RNA Stabilization Reagent. After harvesting, thetissue should be immediately placed in at least 10 volumes of the reagent (orapproximately 10 µl reagent per 1 mg tissue). Larger volumes can be used if necessaryor desired. Smaller volumes may lead to RNA degradation during storage. Storagecontainers should be wide enough so that the reagent covers the entire tissue. Storagecontainers or tubes with large diameters may require more reagent to completely coverthe tissue. The procedures for tissue harvesting and RNA stabilization should be carriedout as quickly as possible.Tissue size is critical for successful RNA stabilization with RNAlater RNA StabilizationReagent. Immediately upon contact, the reagent diffuses into the surface layer and outerportions of solid tissues. To ensure rapid and reliable stabilization of RNA even in theinner parts of solid tissues, the sample must be cut into slices less than 0.5 cm thick. Theslices can be any convenient size, provided one dimension of the sample is <0.5 cm.If the slices are thicker than 0.5 cm, the reagent will diffuse too slowly into the interiorof the sample and RNA degradation will occur. Small organs such as rat kidney andspleen or most mouse organs (except liver) do not require slicing: the entire organ canbe placed in RNAlater RNA Stabilization Reagent.The following guide may help you to determine the amount of RNAlater RNAStabilization Reagent required for RNA stabilization:

A cube of rat kidney with a 5 mm edge length ([5 mm]3 = 125 mm3 = 125 µl)weighs 150–175 mg and requires at least 1.5–1.75 ml of the reagent.A 3 mm cube ([3 mm]3 = 27 mm3 = 27 µl) of most animal tissues weighs 30–35 mgand requires at least 300–350 µl of the reagent.

Although weighing tissues is generally more accurate, RNA in unstabilized tissues willdegrade during weighing. In some cases, however, it may be more convenient toquickly estimate the weight of tissue pieces. Average weights of various entire adultmouse organs and the corresponding amounts of RNAlater RNA Stabilization Reagentto use are given in Table 7.

StabilizationTable 7. Tissue weights and amounts of RNAlater RNA Stabilization Reagent

Amount of RNAlater RNAMouse organ Weight (mg) Stabilization Reagent (ml)Kidney 180–250 ≥2.5Spleen 100–160 ≥1.6Lung 190–210 ≥2.1Heart 100–170 ≥1.7Liver 1000–1800 ≥18

Important points before startingIf using the RNeasy Protect Mini Kit for the first time, read “Important Notes”(page 16).RNAlater RNA Stabilization Reagent may form a precipitate during storage belowroom temperature (15–25°C). Before using the reagent, redissolve the precipitateby heating to 37°C with agitation.Only fresh, unfrozen tissues can be stabilized using RNAlater RNA StabilizationReagent. Previously frozen tissues thaw too slowly in the reagent, preventing thereagent from diffusing into the tissues quickly enough to prevent RNA degradation.

Procedure1. Before excising the tissue sample, estimate the volume (or weight) of the sample to

be stabilized in RNAlater RNA Stabilization Reagent.2. Determine the appropriate volume of RNAlater RNA Stabilization Reagent for

preserving the tissue. At least 10 volumes of the reagent (or approximately 10 µlreagent per 1 mg of tissue) is required. Pipet the correct amount of reagent into anappropriate collection vessel.Note: Be sure to completely submerge the tissue in RNAlater RNA StabilizationReagent. For details, see “Important notes about RNAlater RNA StabilizationReagent”, above.

3. Excise the tissue sample from the animal and, if necessary, cut it into slices lessthan 0.5 cm thick. Perform this step as quickly as possible and proceedimmediately to step 4.Note: For effective RNA stabilization, the tissue sample must be less than 0.5 cmthick. For details, see “Important notes about RNAlater RNA StabilizationReagent”, above.


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4. Completely submerge the tissue piece(s) in the collection vessel containingRNAlater RNA Stabilization Reagent from step 2.Note: The tissue sample must be immediately submerged in RNAlater RNAStabilization Reagent to protect the RNA.

5. Store the tissue submerged in RNAlater RNA Stabilization Reagent for up to4 weeks at 2–8°C, up to 7 days at 15–25°C, or up to 1 day at 37°C.For archival storage at –20°C, first incubate the tissue overnight in the reagent at2–8°C. Then transfer the tissue, in the reagent, to –20°C for storage.For archival storage at –80°C, first incubate the tissue overnight in the reagent at2–8°C. Then remove the tissue from the reagent, and transfer it to –80°C forstorage.Note: Lower temperatures are recommended for longer storage (e.g., 2–8°C forup to 4 weeks instead of 37°C or room temperature (15–25°C); –20°C or –80°Cfor longer storage).Tissues stored in RNAlater RNA Stabilization Reagent at –20°C may not freeze.The low temperature may cause the formation of crystals or a precipitate in thereagent. This will not affect subsequent RNA purification. There is no need toredissolve the precipitate.RNAlater stabilized tissues stored at –20°C or –80°C can be thawed at roomtemperature and frozen again for up to 20 freeze–thaw cycles without affectingRNA quality or yield.If transporting tissue samples in RNAlater RNA Stabilization Reagent, ensure thatthe tissues always remain submerged in the reagent. Either keep the tubes uprightduring transport or fill the tubes completely with RNAlater RNA StabilizationReagent.

6. After storage, continue with “Protocol: Purification of Total RNA from AnimalTissues” (page 37).

AnimalTissues

Protocol: Purification of Total RNA from Animal TissuesThis protocol requires the RNeasy Mini Kit or RNeasy Protect Mini Kit.

Determining the correct amount of starting materialIt is essential to use the correct amount of starting material to obtain optimal RNA yieldand purity. A maximum amount of 30 mg fresh or frozen tissue or 15–20 mg RNAlaterstabilized tissue (which is partially dehydrated) can generally be processed. For mosttissues, the RNA binding capacity of the RNeasy spin column and the lysing capacityof Buffer RLT will not be exceeded by these amounts. Average RNA yields from varioustissues are given in Table 2 (page 17).Some tissues, such as spleen, parts of brain, lung, and thymus are more difficult to lyseor tend to form precipitates during RNA purification. The volume of Buffer RLT may needto be increased to facilitate complete homogenization and to avoid significantlyreduced RNA yields, DNA contamination, or clogging of the RNeasy spin column. Seethe procedure below for details.RNA yields from fibrous tissues, such as skeletal muscle, heart, and skin, may be lowdue to the abundance of contractile proteins, connective tissue, and collagen. Formaximum RNA yields from these tissues, we recommend using the RNeasy FibrousTissue Mini Kit instead. See page 74 for ordering information.Greater RNA yields from fatty tissues, such as brain and adipose tissue, can beachieved using the RNeasy Lipid Tissue Mini Kit, which uses QIAzol Lysis Reagent foroptimal tissue lysis. See page 74 for ordering information.If there is no information about the nature of your starting material, we recommendstarting with no more than 10 mg tissue. Depending on RNA yield and purity, it maybe possible to use up to 30 mg tissue in subsequent preparations.Do not overload the RNeasy spin column, as this will significantly reduce RNA yield andquality.Weighing tissue is the most accurate way to quantitate the amount of starting material.As a guide, a 3 mm cube (27 mm3) of most animal tissues weighs 30–35 mg.

Important points before startingIf using the RNeasy Kit for the first time, read “Important Notes” (page 16).If working with RNA for the first time, read Appendix A (page 61).For optimal results, stabilize harvested tissues immediately in RNAlater RNAStabilization Reagent (see protocol on page 34). Tissues can be stored in thereagent for up to 1 day at 37°C, 7 days at 15–25°C, or 4 weeks at 2–8°C, orarchived at –20°C or –80°C.


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Fresh, frozen, or RNAlater stabilized tissues can be used. Tissues can be stored at–70°C for several months. Flash-freeze tissues in liquid nitrogen, and immediatelytransfer to –70°C. Do not allow tissues to thaw during weighing or handling priorto disruption in Buffer RLT. Homogenized tissue lysates from step 4 can also bestored at –70°C for several months. Incubate frozen lysates at 37°C in a waterbath until completely thawed and salts are dissolved before continuing with step5. Avoid prolonged incubation, which may compromise RNA integrity.If desired, more than 30 mg tissue can be disrupted and homogenized at the startof the procedure (increase the volume of Buffer RLT proportionately). Use a portionof the homogenate corresponding to no more than 30 mg tissue for RNApurification, and store the rest at –80°C.Buffer RLT may form a precipitate upon storage. If necessary, redissolve bywarming, and then place at room temperature (15–25°C).Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore notcompatible with disinfecting reagents containing bleach. See page 6 for safetyinformation.Perform all steps of the procedure at room temperature. During the procedure,work quickly.Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensurethat the centrifuge does not cool below 20°C.

Things to do before startingβ-Mercaptoethanol (β-ME) must be added to Buffer RLT before use. Add 10 µlβ-ME per 1 ml Buffer RLT. Dispense in a fume hood and wear appropriateprotective clothing. Buffer RLT containing β-ME can be stored at room temperature(15–25°C) for up to 1 month.Alternatively, add 20 µl of 2 M dithiothreitol (DTT) per 1 ml Buffer RLT. The stocksolution of 2 M DTT in water should be prepared fresh or frozen in single-usealiquots. Buffer RLT containing DTT can be stored at room temperature for up to1 month.Buffer RPE is supplied as a concentrate. Before using for the first time, add4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a workingsolution.If performing optional on-column DNase digestion, prepare DNase I stock solutionas described in Appendix D (page 67).

AnimalTissues

Procedure1. Excise the tissue sample from the animal or remove it from storage. Remove

RNAlater stabilized tissues from the reagent using forceps. Determine the amountof tissue. Do not use more than 30 mg.Weighing tissue is the most accurate way to determine the amount.Note: If the tissues were stored in RNAlater Reagent at –20°C, be sure to removeany crystals that may have formed.

2. Follow either step 2a or 2b.2a. For RNAlater stabilized tissues:

If using the entire tissue, place it directly into a suitably sized vessel for disruptionand homogenization, and proceed to step 3.If using only a portion of the tissue, cut it on a clean surface. Weigh the piece to beused, and place it into a suitably sized vessel for disruption and homogenization.Proceed to step 3.RNA in RNAlater stabilized tissues is protected during cutting and weighing oftissues at ambient temperature (15–25°C). It is not necessary to cut the tissues onice or dry ice or in a refrigerated room. Remaining tissues can be stored inRNAlater RNA Stabilization Reagent. Previously stabilized tissues can be storedat –80°C without the reagent.

2b. For unstabilized fresh or frozen tissues:If using the entire tissue, place it directly into a suitably sized vessel for disruptionand homogenization, and proceed immediately to step 3.If using only a portion of the tissue, weigh the piece to be used, and place it intoa suitably sized vessel for disruption and homogenization. Proceed immediatelyto step 3.RNA in harvested tissues is not protected until the tissues are treated with RNAlaterRNA Stabilization Reagent, flash-frozen, or disrupted and homogenized in step 3.Frozen tissues should not be allowed to thaw during handling. The relevantprocedures should be carried out as quickly as possible.Note: Remaining fresh tissues can be placed into RNAlater RNA StabilizationReagent to stabilize RNA (see protocol on page 34). However, previously frozentissues thaw too slowly in the reagent, preventing the reagent from diffusing intothe tissues quickly enough to prevent RNA degradation.


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3. Disrupt the tissue and homogenize the lysate in Buffer RLT (do not use more than30 mg tissue) according to step 3a, 3b, 3c, or 3d.See “Disrupting and homogenizing starting material”, pages 18–21, for moredetails on disruption and homogenization.Note: Ensure that β-ME is added to Buffer RLT before use (see “Things to do beforestarting”).After storage in RNAlater RNA Stabilization Reagent, tissues may become slightlyharder than fresh or thawed tissues. Disruption and homogenization usingstandard methods is usually not a problem. For easier disruption andhomogenization, we recommend using 600 µl Buffer RLT.Note: Incomplete homogenization leads to significantly reduced RNA yields andcan cause clogging of the RNeasy spin column. Homogenization with theTissueLyser LT, TissueLyser II, and rotor–stator homogenizers generally results inhigher RNA yields than with other methods.

Table 8. Volumes of Buffer RLT for tissue disruption and homogenization

Amount of starting material (mg) Volume of Buffer RLT (µl)<20 350 or 600*20–30 600

* Use 600 µl Buffer RLT for tissues stabilized in RNAlater RNA Stabilization Reagent or for difficult-to-lysetissues.

3a. Disruption and homogenization using a rotor–stator homogenizer:Place the weighed (fresh, frozen, or RNAlater stabilized) tissue in a suitably sizedvessel. Add the appropriate volume of Buffer RLT (see Table 8). Immediately disruptand homogenize the tissue using a conventional rotor–stator homogenizer until itis uniformly homogeneous (usually 20–40 s). Proceed to step 4.

3b. Disruption using a mortar and pestle followed by homogenization using aQIAshredder homogenizer:Immediately place the weighed (fresh, frozen, or RNAlater stabilized) tissue inliquid nitrogen, and grind thoroughly with a mortar and pestle. Decant tissuepowder and liquid nitrogen into an RNase-free, liquid-nitrogen–cooled, 2 mlmicrocentrifuge tube (not supplied). Allow the liquid nitrogen to evaporate, but donot allow the tissue to thaw.Add the appropriate volume of Buffer RLT (see Table 8). Pipet the lysate directlyinto a QIAshredder spin column placed in a 2 ml collection tube, and centrifuge for2 min at full speed. Proceed to step 4.

AnimalTissues

3c. Disruption using a mortar and pestle followed by homogenization using a needleand syringe:Immediately place the weighed (fresh, frozen, or RNAlater stabilized) tissue inliquid nitrogen, and grind thoroughly with a mortar and pestle. Decant tissuepowder and liquid nitrogen into an RNase-free, liquid-nitrogen–cooled, 2 mlmicrocentrifuge tube (not supplied). Allow the liquid nitrogen to evaporate, but donot allow the tissue to thaw.Add the appropriate volume of Buffer RLT (see Table 8), and homogenize bypassing the lysate at least 5 times through a blunt 20-gauge needle fitted to anRNase-free syringe. Proceed to step 4.

3d. Disruption and homogenization using the TissueLyser LT or TissueLyser II:See the TissueLyser LT Handbook or the TissueLyser Handbook. Then proceed tostep 4.

4. Centrifuge the lysate for 3 min at full speed. Carefully remove the supernatant bypipetting, and transfer it to a new microcentrifuge tube (not supplied). Use only thissupernatant (lysate) in subsequent steps.In some preparations, very small amounts of insoluble material will be present afterthe 3 min centrifugation, making the pellet invisible.

5. Add 1 volume of 70% ethanol* to the cleared lysate, and mix immediately bypipetting. Do not centrifuge. Proceed immediately to step 6.Note: The volume of lysate may be less than 350 µl or 600 µl due to loss duringhomogenization and centrifugation in steps 3 and 4.Note: Precipitates may be visible after addition of ethanol. This does not affect theprocedure.

6. Transfer up to 700 µl of the sample, including any precipitate that may haveformed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Closethe lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard theflow-through.†

Reuse the collection tube in step 7.If the sample volume exceeds 700 µl, centrifuge successive aliquots in the sameRNeasy spin column. Discard the flow-through after each centrifugation.†

Optional: If performing optional on-column DNase digestion (see “Eliminatinggenomic DNA contamination”, page 21), follow steps D1–D4 (page 67) afterperforming this step.

* Using 50% ethanol (instead of 70% ethanol) may increase RNA yields from liver samples.† Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6

for safety information.


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7. Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, andcentrifuge for 15 s at≥8000 x g (≥10,000 rpm) to wash the spin column membrane.Discard the flow-through.*Reuse the collection tube in step 8.Note: After centrifugation, carefully remove the RNeasy spin column from thecollection tube so that the column does not contact the flow-through. Be sure toempty the collection tube completely.Skip this step if performing optional on-column DNase digestion (page 67).







Yeast

Protocol: Purification of Total RNA from YeastThis protocol requires the RNeasy Mini Kit.

Disrupting yeast cellsThis protocol for purifying total RNA from yeast provides 2 alternative methods ofdisrupting the walls of yeast cells:

Enzymatic lysis: This method requires digestion of the cell wall with zymolase orlyticase to convert cells to spheroplasts. For samples of up to 5 x 107 yeast cells,spheroplasts are separated from the digestion mixture by centrifugation beforebeing lysed. For samples of up to 2 x 107 yeast cells, the digestion mixture is useddirectly in the RNeasy procedure without prior separation of the spheroplasts.Mechanical disruption: This method uses high-speed agitation in the TissueLyser LT,TissueLyser II, or other bead mill in the presence of glass beads and Buffer RLT tolyse yeast cells and release RNA.

In general, both methods function equally well. For some applications, enzymatic lysismight be preferable since no additional laboratory equipment is required. Mechanicaldisruption, however, is well-suited for time-course experiments where enzymaticdigestion incubations are not practical.

Determining the correct amount of starting materialIt is essential to use the correct amount of starting material to obtain optimal RNA yieldand purity. The maximum amount depends on:

The RNA binding capacity of the RNeasy spin column (100 µg RNA)The volume of Buffer RLT required for efficient lysis (the maximum volume of BufferRLT that can be used limits the maximum amount of starting material to 5 x 107

yeast cells)When processing cultures containing high amounts of RNA, fewer cells should be used,so that the RNA binding capacity of the RNeasy spin column is not exceeded. Whenprocessing cultures containing lower amounts of RNA, the maximum number of cellscan be used. However, even though the RNA binding capacity of the RNeasy spincolumn is not reached, using more cells would lead to incomplete lysis, resulting in lowerRNA yield and purity.Usually 2 x 106 – 5 x 107 yeast cells can be processed. Depending on the strain andgrowth conditions, 30–100 µg RNA can be expected from 4 x 107 cells.If there is no information about the RNA content of your starting material, werecommend starting with no more than 2 x 107 yeast cells. Depending on RNA yieldand purity, it may be possible to increase the cell number in subsequent preparations.


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Do not overload the RNeasy spin column, as this will significantly reduce RNA yield andpurity.Yeast growth is usually measured using a spectrophotometer. However, it is very difficultto give specific and reliable recommendations for the relationship between OD valuesand cell numbers in yeast cultures. Cell density is influenced by a variety of factors (e.g.,species, media, and shaker speed), and OD readings of cultures measure lightscattering rather than absorption. Measurements of light scattering are highlydependent on the distance between the sample and the detector and therefore readingsvary between different types of spectrophotometer. In addition, different species showdifferent OD values at defined wavelengths (e.g., 600 nm or 436 nm).We therefore recommend calibrating the spectrophotometer by comparing ODmeasurements at appropriate wavelengths with viable cell densities determined byplating experiments (e.g., Ausubel, F.M. et al., eds. [1991] Current Protocols inMolecular Biology. New York: Wiley Interscience). OD readings should be between0.05 and 0.3 to ensure significance. Samples with readings above 0.3 should bediluted so that the readings fall within this range; the dilution factor should then be usedto calculate the number of cells per milliliter.The following values may be used as a rough guide. An S. cerevisiae culture containing1–2 x 107 cells per milliliter, diluted 1 in 4, gives an OD600 value of approximately 0.25with a Beckman DU®-7400 spectrophotometer or 0.125 with a Beckman DU-40spectrophotometer. These correspond to calculated OD values of 1 or 0.5, respectively,for 1–2 x 107 yeast cells per milliliter.

Important points before startingIf using the RNeasy Kit for the first time, read “Important Notes” (page 16).If working with RNA for the first time, read Appendix A (page 61).Yeast cells should be harvested in log-phase growth. If performing enzymatic lysis(step 1a or 1b), use only freshly harvested cells. If performing mechanicaldisruption, cell pellets can be stored at –70°C for later use or used directly in theprocedure. Homogenized cell lysates from step 1c can be stored at –70°C forseveral months. Frozen lysates should be incubated at 37°C in a water bath untilcompletely thawed and salts are dissolved. Avoid prolonged incubation, whichmay compromise RNA integrity. Proceed to step 2.Buffer RLT may form a precipitate upon storage. If necessary, redissolve bywarming, and then place at room temperature (15–25°C).Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore notcompatible with disinfecting reagents containing bleach. See page 6 for safetyinformation.After enzymatic lysis or mechanical disruption, perform all steps of the procedureat room temperature. During the procedure, work quickly.

YeastAfter harvesting the cells, perform all centrifugation steps at 20–25°C in astandard microcentrifuge. Ensure that the centrifuge does not cool below 20°C.

Things to do before startingβ-Mercaptoethanol (β-ME) must be added to Buffer RLT before use. Add 10 µlβ-ME per 1 ml Buffer RLT. Dispense in a fume hood and wear appropriateprotective clothing. Buffer RLT containing β-ME can be stored at room temperature(15–25°C) for up to 1 month.Buffer RPE is supplied as a concentrate. Before using for the first time, add4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a workingsolution.If performing optional on-column DNase digestion, prepare DNase I stock solutionas described in Appendix D (page 67).If performing enzymatic lysis (step 1a or 1b), prepare Buffer Y1 as follows. Preparea solution containing 1 M sorbitol and 0.1 M EDTA, pH 7.4. Just before use, add0.1% β-ME and lyticase/zymolase (final concentration of 50 U per 1 x 107 cells).Depending on the yeast strain and enzyme used, the incubation time, enzymeconcentration, and composition of Buffer Y1 may vary. Please follow the guidelinesof the enzyme supplier.If performing mechanical disruption (step 1c), prepare acid-washed glass beads,0.45–0.55 mm diameter, by soaking in concentrated nitric acid for 1 hour in afume hood, washing extensively with deionized water, and drying in a bakingoven.

Procedure1. Prepare yeast lysate according to step 1a (enzymatic lysis for ≤5 x 107 cells), step

1b (enzymatic lysis for ≤2 x 107 cells), or step 1c (mechanical disruption).1a. Enzymatic lysis of ≤5 x 107 freshly harvested cells (do not use more than 5 x 107

cells):Harvest the cells in a 12 ml or 15 ml centrifuge tube by centrifuging at1000 x g for 5 min at 4°C. Decant the supernatant, and carefully remove anyremaining media by aspiration. If the centrifuge will be used later in thisprocedure, heat it to 20–25°C.Note: Incomplete removal of medium will affect digestion of the cell wall.Resuspend the cells in 2 ml freshly prepared Buffer Y1 containing lyticase orzymolase. Incubate for 10–30 min at 30°C with gentle shaking to generatespheroplasts. Spheroplasts must be handled gently.


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Depending on the yeast strain used, the incubation time, amount of enzyme,and composition of Buffer Y1 may vary. For optimal results, follow theguidelines of the enzyme supplier. Complete spheroplasting is essential forefficient lysis.Centrifuge for 5 min at 300 x g to pellet the spheroplasts. Carefully removeand discard the supernatant.Note: Incomplete removal of the supernatant will inhibit lysis and dilute thelysate, affecting the conditions for binding of RNA to the RNeasy membrane.Both effects may reduce RNA yield.Add 350 µl Buffer RLT and vortex vigorously to lyse the spheroplasts. Ifinsoluble material is visible, centrifuge for 2 min at full speed, and use onlythe supernatant in subsequent steps.Note: Ensure that β-ME is added to Buffer RLT before use (see “Things to dobefore starting”).Add 1 volume (usually 350 µl) of 70% ethanol to the homogenized lysate, andmix well by pipetting. Do not centrifuge. Proceed immediately to step 2.Precipitates may be visible after addition of ethanol. This does not affect theprocedure.

1b. Enzymatic lysis of ≤2 x 107 freshly harvested cells (do not use more than 2 x 107

cells):Harvest the cells in a 12 ml or 15 ml centrifuge tube by centrifuging at1000 x g for 5 min at 4°C. Decant the supernatant, and carefully remove anyremaining media by aspiration. If the centrifuge will be used later in thisprocedure, heat it to 20–25°C.Note: Incomplete removal of medium will affect digestion of the cell wall.Resuspend the cells in 100 µl freshly prepared Buffer Y1 containing lyticaseor zymolase. Incubate for 10–30 min at 30°C with gentle shaking to generatespheroplasts. Spheroplasts must be handled gently.Depending on the yeast strain used, the incubation time, amount of enzyme,and composition of Buffer Y1 may vary. For optimal results, follow theguidelines of the enzyme supplier. Complete spheroplasting is essential forefficient lysis.Add 350 µl Buffer RLT and vortex vigorously to lyse the spheroplasts. Ifinsoluble material is visible, centrifuge for 2 min at full speed, and use onlythe supernatant in subsequent steps.Note: Ensure that β-ME is added to Buffer RLT before use (see “Things to dobefore starting”).Add 250 µl ethanol (96–100%) to the homogenized lysate, and mix well bypipetting. Do not centrifuge. Proceed immediately to step 2.

YeastPrecipitates may be visible after addition of ethanol. This does not affect theprocedure.

1c. Mechanical disruption of cells (do not use more than 5 x 107 cells):Add approximately 600 µl of acid-washed glass beads to a tube that fits theTissueLyser LT, TissueLyser II, or other bead mill (see page 20 for details).Harvest the cells by centrifuging at 1000 x g for 5 min at 4°C. Decant thesupernatant, and carefully remove any remaining media by aspiration. If thecentrifuge will be used later in this procedure, heat it to 20–25°C.Note: Incomplete removal of the supernatant will inhibit lysis and dilute thelysate, affecting the conditions for binding of RNA to the RNeasy membrane.Both effects may reduce RNA yield.Loosen the cell pellet thoroughly by flicking the tube. Add 600 µl Buffer RLT,and vortex to resuspend the cell pellet. Add the sample to the acid-washedglass beads.Note: Ensure that β-ME is added to Buffer RLT before use (see “Things to dobefore starting”).Vortex and agitate the sample at top speed in the TissueLyser LT, TissueLyser II,or other bead mill with cooling until cells are completely disrupted.Most small-capacity bead mills do not have a cooling mechanism andtherefore require the user to stop the bead mill regularly and cool the sampleon ice. The time required for cell disruption and the length and frequency ofthe cooling intervals may vary depending on the type of bead mill used.Please refer to the supplier’s instructions.Note: Do not replace bead-milling with vortexing, as this significantly reducesRNA yield.Remove the sample from the TissueLyser LT, TissueLyser II, or bead mill, andallow the beads to settle. Transfer the lysate (usually 350 µl) to a newmicrocentrifuge tube (not supplied). Centrifuge for 2 min at full speed, andtransfer the supernatant to a new microcentrifuge tube (not supplied). Useonly the supernatant in subsequent steps.Add 1 volume of 70% ethanol to the homogenized lysate, and mix well bypipetting. Do not centrifuge. Proceed to step 2.Note: The volume of lysate may be less than 350 µl due to loss duringhomogenization.Note: Precipitates may be visible after addition of ethanol. This does notaffect the procedure.


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2. Transfer the sample (usually 700 µl), including any precipitate that may haveformed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Closethe lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard theflow-through.*Reuse the collection tube in step 3.If the sample volume exceeds 700 µl, centrifuge successive aliquots in the sameRNeasy spin column. Discard the flow-through after each centrifugation.*Optional: If performing optional on-column DNase digestion (see “Eliminatinggenomic DNA contamination”, page 21), follow steps D1–D4 (page 67) afterperforming this step.

3. Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, andcentrifuge for 15 s at≥8000 x g (≥10,000 rpm) to wash the spin column membrane.Discard the flow-through.*Reuse the collection tube in step 4.Note: After centrifugation, carefully remove the RNeasy spin column from thecollection tube so that the column does not contact the flow-through. Be sure toempty the collection tube completely.Skip this step if performing optional on-column DNase digestion (page 67).

4. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, andcentrifuge for 15 s at≥8000 x g (≥10,000 rpm) to wash the spin column membrane.Discard the the flow-through.Reuse the collection tube in step 5.Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added toBuffer RPE before use (see “Things to do before starting”).



Yeast6. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied),

and discard the old collection tube with the flow-through. Close the lid gently, andcentrifuge at full speed for 1 min.Perform this step to eliminate any possible carryover of Buffer RPE, or if residualflow-through remains on the outside of the RNeasy spin column after step 5.




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Protocol: Purification of Total RNA from Plant Cells andTissues and Filamentous FungiThis protocol requires the RNeasy Plant Mini Kit.

Determining the correct amount of starting materialIt is essential to use the correct amount of starting material to obtain optimal RNA yieldand purity. A maximum amount of 100 mg plant material or 1 x 107 cells can generallybe processed. For most plant materials, the RNA binding capacity of the RNeasy spincolumn and the lysing capacity of Buffer RLT will not be exceeded by these amounts.Average RNA yields from various plant materials are given in Table 2 (page 17).If there is no information about the nature of your starting material, we recommendstarting with no more than 50 mg plant material or 3–4 x 106 cells. Depending on RNAyield and purity, it may be possible to use up to 100 mg plant material or up to 1 x 107

cells in subsequent preparations.Do not overload the RNeasy spin column, as this will significantly reduce RNA yield andquality.Counting cells or weighing tissue is the most accurate way to quantitate the amount ofstarting material. As a guide, a 1.5 cm diameter leaf disc weighs 25–75 mg.

Important points before startingIf using the RNeasy Plant Mini Kit for the first time, read “Important Notes”(page 16).If working with RNA for the first time, read Appendix A (page 61).Fresh or frozen tissues can be used. Tissues can be stored at –70°C for severalmonths. Flash-freeze tissues in liquid nitrogen, and immediately transfer to –70°C.Do not allow tissues to thaw during weighing or handling prior to disruption inBuffer RLT. Homogenized tissue lysates from step 4 can also be stored at –70°Cfor several months. Incubate frozen lysates at 37°C in a water bath until completelythawed and salts are dissolved before continuing with step 5. Avoid prolongedincubation, which may compromise RNA integrity.The RNeasy Plant Mini Kit provides a choice of lysis buffers: Buffer RLT and BufferRLC, which contain guanidine thiocyanate and guanidine hydrochloride,respectively. In most cases, Buffer RLT is the lysis buffer of choice due to the greatercell disruption and denaturation properties of guanidine thiocyanate. However,depending on the amount and type of secondary metabolites in some tissues (suchas milky endosperm of maize or mycelia of filamentous fungi), guanidinethiocyanate can cause solidification of the sample, making extraction of RNAimpossible. In these cases, Buffer RLC should be used.

Plantsand

FungiBuffer RLT may form a precipitate upon storage. If necessary, redissolve bywarming, and then place at room temperature (15–25°C).Buffer RLT, Buffer RLC, and Buffer RW1 contain a guanidine salt and are thereforenot compatible with disinfecting reagents containing bleach. See page 6 for safetyinformation.Perform all steps of the procedure at room temperature. During the procedure,work quickly.Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensurethat the centrifuge does not cool below 20°C.

Things to do before startingβ-Mercaptoethanol (β-ME) must be added to Buffer RLT or Buffer RLC before use.Add 10 µl β-ME per 1 ml Buffer RLT or Buffer RLC. Dispense in a fume hood andwear appropriate protective clothing. Buffer RLT or Buffer RLC containing β-ME canbe stored at room temperature (15–25°C) for up to 1 month.Buffer RPE is supplied as a concentrate. Before using for the first time, add4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a workingsolution.If performing optional on-column DNase digestion, prepare DNase I stock solutionas described in Appendix D (page 67).

Procedure1. Determine the amount of plant material. Do not use more than 100 mg.

Weighing tissue is the most accurate way to determine the amount.2. Immediately place the weighed tissue in liquid nitrogen, and grind thoroughly with

a mortar and pestle. Decant tissue powder and liquid nitrogen into an RNase-free,liquid-nitrogen–cooled, 2 ml microcentrifuge tube (not supplied). Allow the liquidnitrogen to evaporate, but do not allow the tissue to thaw. Proceed immediately tostep 3.RNA in plant tissues is not protected until the tissues are flash-frozen in liquidnitrogen. Frozen tissues should not be allowed to thaw during handling. Therelevant procedures should be carried out as quickly as possible.


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3. Add 450 µl Buffer RLT or Buffer RLC (see “Important points before starting”) to amaximum of 100 mg tissue powder. Vortex vigorously.A short 1–3 min incubation at 56°C may help to disrupt the tissue. However, donot incubate samples with a high starch content at elevated temperatures,otherwise swelling of the sample will occur.Note: Ensure that β-ME is added to Buffer RLT or Buffer RLC before use (see “Thingsto do before starting”).

4. Transfer the lysate to a QIAshredder spin column (lilac) placed in a 2 ml collectiontube, and centrifuge for 2 min at full speed. Carefully transfer the supernatant ofthe flow-through to a new microcentrifuge tube (not supplied) without disturbingthe cell-debris pellet in the collection tube. Use only this supernatant in subsequentsteps.It may be necessary to cut off the end of the pipet tip to facilitate pipetting of thelysate into the QIAshredder spin column. Centrifugation through the QIAshredderspin column removes cell debris and simultaneously homogenizes the lysate.While most of the cell debris is retained on the QIAshredder spin column, a verysmall amount of cell debris will pass through and form a pellet in the collectiontube. Be careful not to disturb this pellet when transferring the lysate to the newmicrocentrifuge tube.

5. Add 0.5 volume of ethanol (96–100%) to the cleared lysate, and mix immediatelyby pipetting. Do not centrifuge. Proceed immediately to step 6.Note: The volume of lysate may be less than 450 µl due to loss duringhomogenization.Note: Precipitates may be visible after addition of ethanol. This does not affect theprocedure.

6. Transfer the sample (usually 650 µl), including any precipitate that may haveformed, to an RNeasy spin column (pink) placed in a 2 ml collection tube (supplied).Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discardthe flow-through.*Reuse the collection tube in step 7.If the sample volume exceeds 700 µl, centrifuge successive aliquots in the sameRNeasy spin column. Discard the flow-through after each centrifugation.*Optional: If performing optional on-column DNase digestion (see “Eliminatinggenomic DNA contamination”, page 21), follow steps D1–D4 (page 67) afterperforming this step.

* Flow-through contains Buffer RLT, Buffer RLC, or Buffer RW1 and is therefore not compatible with bleach.See page 6 for safety information.

Plantsand

Fungi7. Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and

centrifuge for 15 s at≥8000 x g (≥10,000 rpm) to wash the spin column membrane.Discard the flow-through.*Reuse the collection tube in step 8.Note: After centrifugation, carefully remove the RNeasy spin column from thecollection tube so that the column does not contact the flow-through. Be sure toempty the collection tube completely.Skip this step if performing optional on-column DNase digestion (page 67).






* Flow-through contains Buffer RLT, Buffer RLC, or Buffer RW1 and is therefore not compatible with bleach.See page 6 for safety information.


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Protocol: RNA CleanupThe RNeasy Mini Kit can be used to clean up RNA previously isolated by differentmethods or after enzymatic reactions, such as labeling or DNase digestion.

Determining the correct amount of starting materialA maximum of 100 µg RNA can be cleaned up in this protocol. This amountcorresponds to the RNA binding capacity of the RNeasy spin column.

Important points before startingIf using the RNeasy Kit for the first time, read “Important Notes” (page 16).If working with RNA for the first time, read Appendix A (page 61).Buffer RLT may form a precipitate upon storage. If necessary, redissolve bywarming, and then place at room temperature (15–25°C).Buffer RLT contains a guanidine salt and is therefore not compatible withdisinfecting reagents containing bleach. See page 6 for safety information.Perform all steps of the procedure at room temperature. During the procedure,work quickly.Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensurethat the centrifuge does not cool below 20°C.

Things to do before startingBuffer RPE is supplied as a concentrate. Before using for the first time, add4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a workingsolution.If performing optional on-column DNase digestion, prepare DNase I stock solutionas described in Appendix D (page 67).

Procedure1. Adjust the sample to a volume of 100 µl with RNase-free water. Add 350 µl Buffer

RLT, and mix well.2. Add 250 µl ethanol (96–100%) to the diluted RNA, and mix well by pipetting. Do

not centrifuge. Proceed immediately to step 3.

RNA

Cleanup3. Transfer the sample (700 µl) to an RNeasy Mini spin column placed in a 2 ml

collection tube (supplied). Close the lid gently, and centrifuge for 15 s at≥8000 x g (≥10,000 rpm). Discard the flow-through.*Reuse the collection tube in step 4.Note: After centrifugation, carefully remove the RNeasy spin column from thecollection tube so that the column does not contact the flow-through. Be sure toempty the collection tube completely.Optional: If performing optional on-column DNase digestion (see “Eliminatinggenomic DNA contamination”, page 21), follow steps D1–D4 (page 67) afterperforming this step.

4. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, andcentrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane.Discard the flow-through.Reuse the collection tube in step 5.Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added toBuffer RPE before use (see “Things to do before starting”).





* Flow-through contains Buffer RLT and is therefore not compatible with bleach. See page 6 for safety information.


Troubleshooting GuideThis troubleshooting guide may be helpful in solving any problems that may arise. Formore information, see also the Frequently Asked Questions page at our Technical SupportCenter: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN TechnicalServices are always happy to answer any questions you may have about either theinformation and protocols in this handbook or sample and assay technologies (for contactinformation, see back cover or visit www.qiagen.com).

Comments and suggestions

Clogged RNeasy spin columna) Inefficient disruption See “Disrupting and homogenizing starting

and/or homogenization material” (pages 18–21) for details ondisruption and homogenization methods.Increase g-force and centrifugation time ifnecessary.In subsequent preparations, reduce theamount of starting material (see protocols)and/or increase the volume of lysis bufferand the homogenization time.If working with tissues rich in proteins, werecommend using the RNeasy Fibrous TissueMini Kit (see page 74 for orderinginformation).

b) Too much starting material In subsequent preparations, reduce theamount of starting material. It is essential touse the correct amount of starting material(see protocols).

c) Centrifugation before adding Centrifuge the lysate before adding ethanol,ethanol not performed (protocols and use only this supernatant in subsequentfor tissues and mechanical steps (see protocols). Pellets contain celldisruption of yeast) debris that can clog the RNeasy spin column.



d) Centrifugation temperature too low The centrifugation temperature should be20–25°C. Some centrifuges may cool tobelow 20°C even when set at 20°C. Thiscan cause formation of precipitates that canclog the RNeasy spin column. If thishappens, set the centrifugation temperatureto 25°C. Warm the ethanol-containinglysate to 37°C before transferring it to theRNeasy spin column.

Low RNA yielda) Insufficient disruption and See “Disrupting and homogenizing starting

homogenization material” (pages 18–21) for details ondisruption and homogenization methods.Increase g-force and centrifugation time ifnecessary.In subsequent preparations, reduce theamount of starting material (see protocols)and/or increase the volume of lysis bufferand the homogenization time.If working with tissues rich in proteins, werecommend using the RNeasy Fibrous TissueMini Kit (see page 74 for orderinginformation).

b) Too much starting material In subsequent preparations, reduce theamount of starting material. It is essential touse the correct amount of starting material(see protocols).

c) RNA still bound to RNeasy Repeat RNA elution, but incubate thespin column membrane RNeasy spin column on the benchtop for

10 min with RNase-free water beforecentrifuging.



d) Ethanol carryover During the second wash with Buffer RPE, besure to centrifuge at ≥8000 x g (≥10,000rpm) for 2 min at 20–25°C to dry theRNeasy spin column membrane. Aftercentrifugation, carefully remove the columnfrom the collection tube so that the columndoes not contact the flow-through.Otherwise, carryover of ethanol will occur.To eliminate any chance of possible ethanolcarryover, place the RNeasy spin column ina new 2 ml collection tube and perform theoptional 1-min centrifugation step asdescribed in the protocols.

e) Incomplete removal of cell-culture When processing cultured cells, ensuremedium (cell samples) complete removal of the cell-culture medium

after harvesting cells (see protocols).Low A260/A280 valueWater used to dilute RNA for Use 10 mM Tris·Cl, pH 7.5, not RNase-freeA260/A280 measurement water, to dilute the sample before measuring

purity (see Appendix B, page 63).RNA degradeda) Harvested animal tissue not Submerge the tissue in the appropriate

immediately stabilized volume of RNAlater RNA StabilizationReagent immediately after harvesting.

b) Too much animal tissue for Reduce the amount of tissue or increase theproper stabilization amount of RNAlater RNA Stabilization

Reagent used for stabilization (see protocolon page 34).

c) Animal tissue too thick for Cut large samples into slices less thanstabilization 0.5 cm thick for stabilization in RNAlater

RNA Stabilization Reagent.d) Frozen animal tissue used for Use only fresh, unfrozen tissue for

stabilization stabilization in RNAlater RNA StabilizationReagent.

e) Storage duration in RNAlater RNAlater stabilized tissue can be stored forRNA Stabilization Reagent up to 1 day at 37°C, up to 7 days atexceeded 15–25°C, or up to 4 weeks at 2–8°C, and

can be archived at –20°C or –80°C.



f) Inappropriate handling of Ensure that tissue samples are properlystarting material stabilized and stored in RNAlater RNA

Stabilization Reagent.For frozen cell pellets or frozen tissuesamples, ensure that they were flash-frozenimmediately in liquid nitrogen and properlystored at –70°C. Perform the RNeasyprocedure quickly, especially the first fewsteps.See Appendix A (page 61), “Handling andstoring starting material” (page 18), and theRNAlater protocol (page 34).

g) RNase contamination Although all RNeasy buffers have beentested and are guaranteed RNase-free,RNases can be introduced during use. Becertain not to introduce any RNases duringthe RNeasy procedure or later handling. SeeAppendix A (page 61) for general remarkson handling RNA.Do not put RNA samples into a vacuumdryer that has been used in DNApreparations where RNases may have beenused.

DNA contamination in downstream experimentsa) Optimal procedure not used For animal cells, we recommend purifying

(cell samples) cytoplasmic RNA for applications where theabsence of DNA contamination is critical,since intact nuclei are removed at the start ofthe procedure. The protocol can bedownloaded at www.qiagen.com/literature/protocols/RNeasyMini.aspx.

b) No incubation with Buffer RW1 In subsequent preparations, incubate theRNeasy spin column for 5 min at roomtemperature (15–25°C) after addition ofBuffer RW1 and before centrifuging.



c) No DNase treatment Perform optional on-column DNasedigestion using the RNase-Free DNase Set(see Appendix D, page 67) at the pointindicated in the individual protocols.Alternatively, after the RNeasy procedure,DNase digest the RNA eluate. Afterinactivating the DNase by heat treatment,the RNA can be either used directly in thedownstream application without furthertreatment, or repurified using the RNAcleanup protocol (page 54).

RNA does not perform well in downstream experimentsa) Salt carryover during elution Ensure that Buffer RPE is at 20–30°C.

When reusing collection tubes betweenwashing steps, remove residual flow-throughfrom the rim by blotting on clean papertowels.

b) Ethanol carryover During the second wash with Buffer RPE, besure to centrifuge at ≥8000 x g (≥10,000rpm) for 2 min at 20–25°C to dry theRNeasy spin column membrane. Aftercentrifugation, carefully remove the columnfrom the collection tube so that the columndoes not contact the flow-through.Otherwise, carryover of ethanol will occur.To eliminate any chance of possible ethanolcarryover, place the RNeasy spin column ina new 2 ml collection tube and perform theoptional 1-min centrifugation step asdescribed in the protocols.


Appendix A: General Remarks on Handling RNA

Handling RNARibonucleases (RNases) are very stable and active enzymes that generally do notrequire cofactors to function. Since RNases are difficult to inactivate and even minuteamounts are sufficient to destroy RNA, do not use any plasticware or glassware withoutfirst eliminating possible RNase contamination. Great care should be taken to avoidinadvertently introducing RNases into the RNA sample during or after the purificationprocedure. To create and maintain an RNase-free environment, the following precautionsmust be taken during pretreatment and use of disposable and nondisposable vesselsand solutions while working with RNA.

General handlingProper microbiological, aseptic technique should always be used when working withRNA. Hands and dust particles may carry bacteria and molds and are the most commonsources of RNase contamination. Always wear latex or vinyl gloves while handlingreagents and RNA samples to prevent RNase contamination from the surface of the skinor from dusty laboratory equipment. Change gloves frequently and keep tubes closedwhenever possible. Keep purified RNA on ice when aliquots are pipetted fordownstream applications.To remove RNase contamination from bench surfaces, nondisposable plasticware, andlaboratory equipment (e.g., pipets and electrophoresis tanks), use of RNaseKiller (cat. no2500080) from 5 PRIME (www.5prime.com) is recommended. RNase contaminationcan alternatively be removed using general laboratory reagents. To decontaminateplasticware, rinse with 0.1 M NaOH, 1 mM EDTA* followed by RNase-free water (see"Solutions", page 62), or rinse with chloroform* if the plasticware is chloroform-resist-ant. To decontaminate electrophoresis tanks, clean with detergent (e.g., 0.5% SDS),*rinse with RNase-free water, rinse with ethanol (if the tanks are ethanol-resistant), andallow to dry.

Disposable plasticwareThe use of sterile, disposable polypropylene tubes is recommended throughout theprocedure. These tubes are generally RNase-free and do not require pretreatment toinactivate RNases.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.


GlasswareGlassware should be treated before use to ensure that it is RNase-free. Glassware usedfor RNA work should be cleaned with a detergent,* thoroughly rinsed, and oven bakedat 240°C for at least 4 hours (overnight, if more convenient) before use. Autoclavingalone will not fully inactivate many RNases. Alternatively, glassware can be treated withDEPC* (diethyl pyrocarbonate), as described in "Solutions" below.

SolutionsSolutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is astrong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of0.1% to inactivate RNases on glass or plasticware or to create RNase-free solutions andwater. DEPC inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 mlof the solution to be treated and shake vigorously to bring the DEPC into solution. Letthe solution incubate for 12 hours at 37°C. Autoclave for 15 minutes to remove anytrace of DEPC. DEPC will react with primary amines and cannot be used directly to treatTris* buffers. DEPC is highly unstable in the presence of Tris buffers and decomposesrapidly into ethanol and CO2. When preparing Tris buffers, treat water with DEPC first,and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC willmodify purine residues in RNA by carbethoxylation. Carbethoxylated RNA is translatedwith very low efficiency in cell-free systems. However, its ability to form DNA:RNA orRNA:RNA hybrids is not seriously affected unless a large fraction of the purine residueshave been modified. Residual DEPC must always be eliminated from solutions or vesselsby autoclaving or heating to 100°C for 15 minutes.Note: RNeasy buffers are guaranteed RNase-free without using DEPC treatment and aretherefore free of any DEPC contamination.



Appendix B: Storage, Quantification, andDetermination of Quality of RNA

Storage of RNAPurified RNA may be stored at –20°C or –70°C in RNase-free water. Under theseconditions, no degradation of RNA is detectable after 1 year.

Quantification of RNAThe concentration of RNA should be determined by measuring the absorbance at260 nm (A260) in a spectrophotometer (see “Spectrophotometric quantification of RNA”below). For small amounts of RNA, however, it may be difficult to determine amountsphotometrically. Small amounts of RNA can be accurately quantified using an Agilent®2100 Bioanalyzer®, quantitative RT-PCR, or fluorometric quantification.

Spectrophotometric quantification of RNATo ensure significance, A260 readings should be greater than 0.15. An absorbance of1 unit at 260 nm corresponds to 44 µg of RNA per ml (A260=1 → 44 µg/ml). Thisrelation is valid only for measurements at a neutral pH. Therefore, if it is necessary todilute the RNA sample, this should be done in a buffer with neutral pH.* As discussedbelow (see “Purity of RNA”, page 64), the ratio between the absorbance values at 260and 280 nm gives an estimate of RNA purity.When measuring RNA samples, be certain that cuvettes are RNase-free, especially ifthe RNA is to be recovered after spectrophotometry. This can be accomplished bywashing cuvettes with 0.1 M NaOH, 1 mM EDTA,* followed by washing with RNase-free water (see “Solutions”, page 62). Use the buffer in which the RNA is diluted to zerothe spectrophotometer. An example of the calculation involved in RNA quantification isshown below:

Volume of RNA sample = 100 µlDilution = 10 µl of RNA sample + 490 µl of 10 mM Tris·Cl,* pH 7.0 (1/50dilution)Measure absorbance of diluted sample in a 1 ml cuvette (RNase-free)A260 = 0.2Concentration of RNA sample = 44 µg/ml x A260 x dilution factor

= 44 µg/ml x 0.2 x 50= 440 µg/ml



Total amount = concentration x volume in milliliters= 440 µg/ml x 0.1 ml= 44 µg of RNA

Purity of RNAThe ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate ofthe purity of RNA with respect to contaminants that absorb in the UV spectrum, such asprotein. However, the A260/A280 ratio is influenced considerably by pH. Since water isnot buffered, the pH and the resulting A260/A280 ratio can vary greatly. Lower pH resultsin a lower A260/A280 ratio and reduced sensitivity to protein contamination.* Foraccurate values, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. PureRNA has an A260/A280 ratio of 1.9–2.1† in 10 mM Tris·Cl, pH 7.5. Always be sure tocalibrate the spectrophotometer with the same solution used for dilution.For determination of RNA concentration, however, we recommend dilution of thesample in a buffer with neutral pH since the relationship between absorbance andconcentration (A260 reading of 1 = 44 µg/ml RNA) is based on an extinction coefficientcalculated for RNA at neutral pH (see “Spectrophotometric quantification of RNA”,page 63).

DNA contaminationNo currently available purification method can guarantee that RNA is completely freeof DNA, even when it is not visible on an agarose gel. While RNeasy Kits will removethe vast majority of cellular DNA, trace amounts may still remain, depending on theamount and nature of the sample.For analysis of very low-abundance targets, any interference by residual DNAcontamination can be detected by performing real-time RT-PCR control experiments inwhich no reverse transcriptase is added prior to the PCR step. To prevent any interfer-ence by DNA in gene expression analysis real-time RTPCR applications, such as withApplied Biosystems® and Rotor-Gene® instruments, we recommend designing primers thatanneal at intron splice junctions so that genomic DNA will not be amplified. QuantiTectPrimer Assays from QIAGEN are designed for SYBR Green based real-time RT-PCRanalysis of RNA sequences (without detection of genomic DNA) where possible (theassays can be ordered online at www.qiagen.com/GeneGlobe). For real-time RT-PCRassays where amplification of genomic DNA cannot be avoided, we recommend usingthe QuantiTect Reverse Transcription Kit for reverse transcription. The kit integrates fastcDNA synthesis with rapid removal of genomic DNA contamination.

* Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on thespectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.

† Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with somespectrophotometers.


Alternatively, gene expression analysis can be performed using QuantiFast® ProbeAssays and the QuantiFast Probe RT-PCR Plus Kit, which includes an integrated genomicDNA removal step.The protocol for purification of cytoplasmic RNA from animal cells (available atwww.qiagen.com/literature/protocols/RNeasyMini.aspx) is particularly advantageousin applications where the absence of DNA contamination is critical, since intact nucleiare removed. Using this protocol, DNase digestion is generally not required: most ofthe DNA is removed with the nuclei, and RNeasy technology efficiently removes nearlyall of the remaining small amounts of DNA without DNase treatment. However, evenfurther DNA removal may be desirable for certain RNA applications that are sensitiveto very small amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundancetarget). Using the cytoplasmic RNA protocol with optional DNase digestion results inundetectable levels of DNA, even in sensitive quantitative RT-PCR analyses.

Integrity of RNAThe integrity and size distribution of total RNA purified with RNeasy Kits can be checkedby denaturing agarose gel electrophoresis and ethidium bromide* staining or by usingan Agilent 2100 bioanalyzer. The respective ribosomal RNAs should appear as sharpbands or peaks. The apparent ratio of 28S rRNA to 18S RNA should be approximately2:1. If the ribosomal bands or peaks of a specific sample are not sharp, but appear asa smear towards smaller sized RNAs, it is likely that the sample suffered majordegradation either before or during RNA purification.

Appendix C: Formaldehyde Agarose GelElectrophoresisThe following protocol for formaldehyde agarose (FA) gel electrophoresis is routinelyused at QIAGEN and gives enhanced sensitivity for gel and subsequent analysis (e.g.,northern blotting). A key feature is the concentrated RNA loading buffer that allows alarger volume of RNA sample to be loaded onto the gel than conventional protocols(e.g., Sambrook, J. et al. [1989] Molecular cloning — a laboratory manual. 2nd ed.Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).



FA gel preparationTo prepare FA gel (1.2% agarose) of size 10 x 14 x 0.7 cm, mix:

1.2 g agarose*10 ml 10x FA gel buffer (see composition below)Add RNase-free water to 100 ml

If smaller or larger gels are needed, adjust the quantities of components proportionately.Heat the mixture to melt agarose. Cool to 65°C in a water bath. Add 1.8 ml of 37%(12.3 M) formaldehyde* and 1 µl of a 10 mg/ml ethidium bromide* stock solution.Mix thoroughly and pour onto gel support. Prior to running the gel, equilibrate in 1x FAgel running buffer (see composition below) for at least 30 min.

RNA sample preparation for FA gel electrophoresisAdd 1 volume of 5x RNA loading buffer (see composition below) to 4 volumes of RNAsample (e.g., 10 µl of loading buffer and 40 µl of RNA) and mix.Incubate for 3–5 min at 65°C, chill on ice, and load onto the equilibrated FA gel.

Gel running conditionsRun gel at 5–7 V/cm in 1x FA gel running buffer.

Composition of FA gel buffers10x FA gel buffer

200 mM 3-[N-morpholino]propanesulfonic acid (MOPS) (free acid)*50 mM sodium acetate*10 mM EDTA*pH to 7.0 with NaOH*

1x FA gel running buffer100 ml 10x FA gel buffer20 ml 37% (12.3 M) formaldehyde880 ml RNase-free water



5x RNA loading buffer16 µl saturated aqueous bromophenol blue solution*†

80 µl 500 mM EDTA, pH 8.0720 µl 37% (12.3 M) formaldehyde2 ml 100% glycerol*3.084 ml formamide*4 ml 10 x FA gel bufferRNase-free water to 10 mlStability: approximately 3 months at 4°C

Appendix D: Optional On-Column DNase Digestionwith the RNase-Free DNase SetThe RNase-Free DNase Set (cat. no. 79254) provides efficient on-column digestion ofDNA during RNA purification. The DNase is efficiently removed in subsequent washsteps.Note: Standard DNase buffers are not compatible with on-column DNase digestion.Use of other buffers may affect the binding of RNA to the RNeasy membrane, reducingRNA yield and integrity.Lysis and homogenization of the sample and binding of RNA to the RNeasy membraneare performed according to the standard protocols. After washing with a reducedvolume of Buffer RW1, the RNA is treated with DNase I while bound to the RNeasymembrane. The DNase I is removed by a second wash with Buffer RW1. Washing withBuffer RPE and elution of RNA are then performed according to the standard protocols.

Important points before startingGenerally, DNase digestion is not required since RNeasy technology efficientlyremoves most of the DNA without DNase treatment. However, further DNAremoval may be necessary for certain RNA applications that are sensitive to verysmall amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundant target).DNA can also be removed by a DNase digestion following RNA purification.Do not vortex the reconstituted DNase I. DNase I is especially sensitive to physicaldenaturation. Mixing should only be carried out by gently inverting the tube.


† To make a saturated solution, add solid bromophenol blue to distilled water. Mix and continue to add morebromophenol blue until no more will dissolve. Centrifuge to pellet the undissolved powder, and carefullypipet the saturated supernatant.


Things to do before startingPrepare DNase I stock solution before using the RNase-Free DNase Set for the firsttime. Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNase-free water provided. To avoid loss of DNase I, do not open the vial. Inject RNase-free water into the vial using an RNase-free needle and syringe. Mix gently byinverting the vial. Do not vortex.For long-term storage of DNase I, remove the stock solution from the glass vial,divide it into single-use aliquots, and store at –20°C for up to 9 months. Thawedaliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquotsafter thawing.

ProcedurePrepare and load samples onto the RNeasy spin column as indicated in the individualprotocols. Instead of performing the first wash step, follow steps D1–D4 below.D1. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and

centrifuge for 15 s at≥8000 x g (≥10,000 rpm) to wash the spin column membrane.Discard the flow-through.*Reuse the collection tube in step D4.

D2. Add 10 µl DNase I stock solution (see above) to 70 µl Buffer RDD. Mix by gentlyinverting the tube, and centrifuge briefly to collect residual liquid from the sides ofthe tube.Buffer RDD is supplied with the RNase-Free DNase Set.Note: DNase I is especially sensitive to physical denaturation. Mixing should onlybe carried out by gently inverting the tube. Do not vortex.

D3. Add the DNase I incubation mix (80 µl) directly to the RNeasy spin columnmembrane, and place on the benchtop (20–30°C) for 15 min.Note: Be sure to add the DNase I incubation mix directly to the RNeasy spincolumn membrane. DNase digestion will be incomplete if part of the mix sticks tothe walls or the O-ring of the spin column.

D4. Add 350 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, andcentrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.*Continue with the first Buffer RPE wash step in the relevant protocol.Note: In most of the protocols, the immediately following Buffer RW1 wash step isskipped (as indicated in the protocol). Continue with the first Buffer RPE wash step.

* Flow-through contains Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety information.


Appendix E: DNase Digestion of RNA before RNACleanupThis protocol describes how to use the RNase-Free DNase Set (cat. no. 79254) to digestcontaminating DNA in RNA solutions prior to RNA cleanup. DNase digestion canalternatively be carried out during RNA cleanup (see Appendix D, page 67). Forsamples highly contaminated with DNA, we recommend DNase digestion in solution,as it is more efficient than on-column DNase digestion.

Important points before startingGenerally, DNase digestion is not required since RNeasy technology efficientlyremoves most of the DNA without DNase treatment. However, further DNAremoval may be necessary for certain RNA applications that are sensitive to verysmall amounts of DNA (e.g., TaqMan RT-PCR analysis with a low-abundant target).Do not vortex the reconstituted DNase I. DNase I is especially sensitive to physicaldenaturation. Mixing should only be carried out by gently inverting the tube.

Things to do before startingPrepare DNase I stock solution before using the RNase-Free DNase Set for the firsttime. Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNase-free water provided. To avoid loss of DNase I, do not open the vial. Inject RNase-free water into the vial using an RNase-free needle and syringe. Mix gently byinverting the vial. Do not vortex.For long-term storage of DNase I, remove the stock solution from the glass vial,divide it into single-use aliquots, and store at –20°C for up to 9 months. Thawedaliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquotsafter thawing.

ProcedureE1. Mix the following in a microcentrifuge tube:

≤87.5 µl RNA solution (contaminated with genomic DNA)10 µl Buffer RDD2.5 µl DNase I stock solution

Make the volume up to 100 µl with RNase-free water.The reaction volumes can be doubled if necessary (to 200 µl final volume).

E2. Incubate on the benchtop (20–25°C) for 10 min.E3. Clean up the RNA according to “Protocol: RNA Cleanup” on page 54.


Appendix F: Acetone Precipitation of Protein fromBuffer RLT LysatesThis protocol is designed for acetone precipitation of protein from cell lysates preparedusing Buffer RLT. The precipitated, denatured protein is suitable for applications such asSDS-PAGE, western blotting, and 2D gel electrophoresis.

Equipment and reagents to be supplied by user*IceBenchtop centrifugeAcetoneOptional: EthanolBuffer for downstream application (e.g., loading buffer for SDS-PAGE gel)

Important point before startingDO NOT use trichloroacetic acid (TCA) to precipitate protein from Buffer RLT lysates.This buffer contains guanidine thiocyanate, which can form highly reactivecompounds when combined with acidic solutions.

ProcedureF1. Prepare cell lysate and centrifuge it through an RNeasy spin column, as described

in the protocols in this handbook.F2. Add 4 volumes of ice-cold acetone to the flow-through from the RNeasy spin

column.F3. Incubate for 30 min on ice or at –20°C.F4. Centrifuge for 10 min at maximum speed in a benchtop centrifuge. Discard the

supernatant and air-dry the pellet.†

F5. Optional: Wash the pellet with 100 µl ice-cold ethanol and air-dry.Do not overdry the pellet as this may make resuspension more difficult.

F6. Resuspend the pellet in the buffer for your downstream application.Sodium dodecyl sulfate (SDS) causes guanidine salts to precipitate. In case thepellet contains traces of guanidine thiocyanate, load the sample onto anSDS-PAGE gel immediately after heating for 7 minutes at 95°C.


† Supernatant contains guanidine thiocyanate and is therefore not compatible with bleach. See page 6 forsafety information.


Appendix G: RT-PCR and Real-Time RT-PCR

RT-PCRTo perform PCR using RNA as a starting template, the RNA must first be reversetranscribed into cDNA in a reverse transcription (RT) reaction. RT and PCR can becarried out either sequentially in the same tube (one-step RT-PCR) or separately (two-stepRT-PCR).One-step RT-PCR requires gene-specific primers. For this application, QIAGEN offers theQIAGEN OneStep RT-PCR Kit, which enables one-step RT-PCR of any RNA templatewithout optimization.Two-step RT-PCR is generally carried out using oligo-dT and ramdom primers in the RTstep and gene-specific primers in the PCR step. For the RT step, QIAGEN offers theQuantiTect Reverse Transcription Kit for efficient and sensitive reverse transcription. Forthe PCR step, QIAGEN offers enzymes that minimize PCR optimization:

Taq DNA Polymerase — for PCR without a hot startHotStarTaq® DNA Polymerase — for PCR with a hot startHotStarTaq Plus DNA Polymerase — for PCR with a hot start and a fast 5-minenzyme activation time

For more information on QIAGEN products for one-step RT-PCR and two-step RT-PCR,visit www.qiagen.com/products/pcr.

Real-time RT-PCRThe range of QuantiTect Kits and Assays guarantee highly specific and sensitive resultsin real-time RT-PCR on any real-time cycler and require no optimization of reaction andcycling conditions. QuantiTect Kits are available for two-step and one-step RT-PCR andare compatible with detection by SYBR® Green I dye or by sequence-specific probes(e.g., TaqMan and FRET probes). Multiplex RT-PCR of up to 4 targets is also possible.Predesigned, validated QuantiTect Assays are supplied as primer sets or primer–probesets and are easily ordered online at www.qiagen.com/GeneGlobe. For moreinformation on QuantiTect Kits and Assays, visit www.qiagen.com/geneXpression.


Ordering Information

Product Contents Cat. No.

RNeasy Mini Kit (50)* 50 RNeasy Mini Spin Columns, 74104Collection Tubes, RNase-FreeReagents and Buffers

RNeasy Mini Kit (250)* 250 RNeasy Mini Spin Columns, 74106Collection Tubes, RNase-FreeReagents and Buffers

RNeasy Protect Mini Kit (50)* 50 ml RNAlater RNA Stabilization 74124Reagent, 50 RNeasy Mini SpinColumns, Collection Tubes,RNase-Free Reagents and Buffers

RNeasy Protect Mini Kit (250)* 250 ml RNAlater RNA Stabilization 74126Reagent, 250 RNeasy Mini SpinColumns, Collection Tubes,RNase-Free Reagents and Buffers

RNeasy Plant Mini Kit (20) 20 RNeasy Mini Spin Columns, 7490320 QIAshredder Mini Spin Columns,Collection Tubes, RNase-FreeReagents and Buffers

RNeasy Plant Mini Kit (50) 50 RNeasy Mini Spin Columns, 7490450 QIAshredder Mini Spin Columns,Collection Tubes, RNase-FreeReagents and Buffers

AccessoriesBuffer RLT (220 ml) 220 ml Buffer RLT 79216Collection Tubes (2 ml) 1000 x 2 ml Collection Tubes 19201RNAlater RNA Stabilization For stabilization of RNA in 76104Reagent (50 ml)† 25 x 200 mg tissue samples:

50 ml RNAlater RNA StabilizationReagent

* Kit also available in midi and maxi formats for larger sample sizes; see www.qiagen.com/RNA.† Other kit sizes are available; see www.qiagen.com.




RNAlater TissueProtect For stabilization of RNA in 76154Tubes (50 x 1.5 ml) 50 x 150 mg tissue samples:

50 screw-top tubes containing1.5 ml RNAlater RNA StabilizationReagent each

RNAlater TissueProtect For stabilization of RNA in 76163Tubes (20 x 5 ml) 20 x 500 mg tissue samples:

20 screw-top tubes containing5 ml RNAlater RNA StabilizationReagent each

Allprotect Tissue Reagent (100 ml) 100 ml Allprotect Tissue Reagent, 76405Allprotect Reagent Pump

RNAprotect Bacteria Reagent RNAprotect Bacteria Reagent 76506(2 x 100 ml)

QIAvac 24 Plus Vacuum manifold for processing 194131–24 spin columns: includesQIAvac 24 Plus Vacuum Manifold,Luer Plugs, Quick Couplings

Vacuum Pump (110 V, 60 Hz) Universal vacuum pump (capacity 8400034 L/min, 8 mbar vacuum abs.)



Vacuum Regulator For use with QIAvac manifolds 19530VacConnectors (500) 500 disposable connectors for use 19407

with QIAGEN spin columns on luerconnectors

QIAshredder (50)* 50 disposable cell-lysate 79654homogenizers

* Other kit sizes are available; see www.qiagen.com.




TissueRuptor Handheld rotor–stator homogenizer, 9001271(120 V, 60 Hz, US/JP) 120 V, 60 Hz (for North America

and Japan), 5 TissueRuptorDisposable Probes

TissueRuptor Disposable Probes 25 nonsterile plastic disposable 990890(25) probes for use with the TissueRuptorTissueLyser II Bead mill, 100-120/220-240 V, 85300

50/60 Hz; requires the TissueLyserAdapter Set 2 x 24 or TissueLyserAdapter Set 2 x 96 (availableseparately)*

TissueLyser LT Compact bead mill, 100-240 V AC, 8560050-60 Hz; requires the TissueLyser LTAdapter, 12-Tube (availableseparately)†

RNase-Free DNase Set (50) For 50 RNA minipreps: 1500 units 79254RNase-Free DNase I, RNase-FreeBuffer RDD, and RNase-Free Water

Related products for RNA purificationRNeasy Plus Universal Kit — for purification of total RNA fromall types of tissue using gDNA Eliminator SolutionRNeasy Plus Universal For 10 RNA midipreps: RNeasy Midi 73442Midi Kit (10)‡ Spin Columns, gDNA Eliminator

Solution, Collection Tubes,RNase-Free Water and Buffers

RNeasy Fibrous Tissue Kit — for purification of total RNA fromfiber-rich tissuesRNeasy Fibrous Tissue 50 RNeasy Mini Spin Columns, 74704Mini Kit (50)‡ Collection Tubes, Proteinase K,

RNase-Free DNase I, RNase-FreeReagents and Buffers

* The TissueLyser II must be used in combination with the TissueLyser Adapter Set2 x 24 or TissueLyser Adapter Set 2 x 96.

† The TissueLyser LT must be used in combination with the TissueLyser LT Adapter, 12-Tube.‡ Other kit sizes are available; see www.qiagen.com.




RNeasy Lipid Tissue Kit — for purification of total RNA from fattytissues (and from other types of tissue)RNeasy Lipid Tissue 50 RNeasy Mini Spin Columns, 74804Mini Kit (50)* Collection Tubes, QIAzol Lysis

Reagent, RNase-Free Reagents andBuffers

RNeasy Protect Bacteria Kit — for in vivo stabilization of the geneexpression profile in bacteria and subsequent RNA purificationRNeasy Protect Bacteria RNeasy Mini Kit (50) and 74524Mini Kit (50)* RNAprotect Bacteria Reagent

(2 x 100 ml)RNeasy Plus Mini Kit — for purification of total RNA from cultured cellsand tissues using gDNA Eliminator columnsRNeasy Plus Mini Kit (50) 50 RNeasy Mini Spin Columns, 74134

50 gDNA Eliminator Mini SpinColumns, Collection Tubes,RNase-Free Reagents and Buffers

RNeasy Micro Kit — for purification of concentrated total RNA fromsmall amounts of tissue or small numbers of cellsRNeasy Micro Kit (50) 50 RNeasy MinElute® Spin Columns, 74004

Collection Tubes, RNase-FreeDNase I, Carrier RNA, RNase-FreeReagents and Buffers

RNeasy MinElute Cleanup Kit — for RNA cleanup and concentrationwith small elution volumesRNeasy MinElute Cleanup 50 RNeasy MinElute Spin Columns, 74204Kit (50) Collection Tubes, RNase-Free

Reagents and Buffers

* Other kit sizes are available; see www.qiagen.com.




Related products for downstream applicationsQIAGEN OneStep RT-PCR Kit — for fast and successful one-step RT-PCRQIAGEN OneStep For 25 x 50 µl reactions: QIAGEN 210210RT-PCR Kit (25)* OneStep RT-PCR Enzyme Mix,

5x QIAGEN OneStep RT-PCR Buffer,dNTP Mix, 5x Q-Solution®,RNase-Free Water

QuantiTect Reverse Transcription Kit — for fast cDNA synthesis forsensitive real-time two-step RT-PCRQuantiTect Reverse Transcription For 50 x 20 µl reactions: gDNA 205311Kit (50)* Wipeout Buffer, Quantiscript®

Reverse Transcriptase, QuantiscriptRT Buffer, RT Primer Mix,RNase-Free Water

* Other kit size available; see www.qiagen.com/products/pcr.




QuantiTect SYBR Green PCR Kit — for quantitative, real-time, two-stepRT-PCR using SYBR Green IQuantiTect SYBR Green PCR For 200 x 50 µl reactions: 204143Kit (200)*† 3 x 1.7 ml 2x Master Mix, 2 x 2 ml

RNase-Free WaterQuantiTect SYBR Green RT-PCR Kit — for quantitative, real-time,one-step RT-PCR using SYBR Green IQuantiTect SYBR Green RT-PCR For 200 x 50 µl reactions: 204243Kit (200)*† 3 x 1.7 ml 2x Master Mix, 100 µl

RT Mix, 2 x 2 ml RNase-Free WaterQuantiTect Probe PCR Kit — for quantitative, real-time, two-stepRT-PCR using sequence-specific probesQuantiTect Probe PCR For 200 x 50 µl reactions: 204343Kit (200)*† 3 x 1.7 ml 2x Master Mix, 2 x 2 ml

RNase-Free WaterQuantiTect Probe RT-PCR Kit — for quantitative, real-time, one-stepRT-PCR using sequence-specific probesQuantiTect Probe RT-PCR For 200 x 50 µl reactions: 204443Kit (200)*† 3 x 1.7 ml 2x Master Mix, 100 µl

RT Mix, 2 x 2 ml RNase-Free Water

* Larger kit size available; see www.qiagen.com/products/pcr.† Visit www.qiagen.com/GeneGlobe to search for and order primer sets or primer–probe sets.





QuantiTect Multiplex PCR Kits — for quantitative, multiplex, real-time,two-step RT-PCR using sequence-specific probesQuantiTect Multiplex PCR For 200 x 50 µl reactions: 204543Kit (200)*†‡ 3 x 1.7 ml 2x Master Mix

(contains ROX dye), 2 x 2 mlRNase-Free Water

QuantiTect Multiplex For 200 x 50 µl reactions: 204743PCR NoROX Kit (200)*†§ 3 x 1.7 ml 2x Master Mix

(contains no ROX dye),2 x 2 ml RNase-Free Water

QuantiTect Multiplex RT-PCR Kits — for quantitative, multiplex,real-time, one-step RT-PCR using sequence-specific probesQuantiTect Multiplex RT-PCR For 200 x 50 µl reactions: 204643Kit (200)*†‡ 3 x 1.7 ml 2x Master Mix

(contains ROX dye), 100 µl RT Mix,2 x 2 ml RNase-Free Water

QuantiTect Multiplex RT-PCR NR For 200 x 50 µl reactions: 204843Kit (200)*†§ 3 x 1.7 ml 2x Master Mix

(contains no ROX dye), 100 µl RTMix, 2 x 2 ml RNase-Free Water

For up-to-date licensing information and product-specific disclaimers, see the respectiveQIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals areavailable at www.qiagen.com or can be requested from QIAGEN Technical Servicesor your local distributor.

* Larger kit size available; see www.qiagen.com/products/pcr.† Visit www.qiagen.com/GeneGlobe to search for and order primer–probe sets.‡ Recommended for ABI PRISM® and Applied Biosystems cyclers.§ Recommended for all other cyclers.

Trademarks:

QIAGEN®, QIAcube®, GeneGlobe®, HotStarTaq®, MinElute®, Q-Solution®, QuantiFast®, Quantiscript®, QuantiTect®,Rotor-Gene®, RNAprotect®, RNeasy® (QIAGEN Group); ABI PRISM® (Applera Corporation or its subsidiaries);Agilent®, Bioanalyzer®, (Agilent Technologies, Inc.); Applied Biosystems® (Applied Biosystems LCC); DU®

(Beckman Coulter, Inc.); LightCycler®, TaqMan® (Roche Group); Polytron® (Kinematica AG); SYBR® (MolecularProbes, Inc.); Tissue-Tearor™ (BioSpec Products, Inc.); Ultra Turrax® (IKA-Werke GmbH & Co. KG).“RNAlater®” is a trademark of AMBION, Inc., Austin, Texas and is covered by various U.S. and foreign patents.Limited License Agreement for RNeasy Mini Kit and RNeasy Protect Mini Kit

Use of this product signifies the agreement of any purchaser or user of the product to the following terms:1. The product may be used solely in accordance with the protocols provided with the product and this handbook

and for use with components contained in the kit only. QIAGEN grants no license under any of its intellectualproperty to use or incorporate the enclosed components of this kit with any components not included withinthis kit except as described in the protocols provided with the product, this handbook, and additional protocolsavailable at www.qiagen.com. Some of these additional protocols have been provided by QIAGEN usersfor QIAGEN users. These protocols have not been thoroughly tested or optimized by QIAGEN. QIAGENneither guarantees them nor warrants that they do not infringe the rights of third-parties.

2. Other than expressly stated licenses, QIAGEN makes no warranty that this kit and/or its use(s) do notinfringe the rights of third-parties.

3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead

to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited LicenseAgreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, inany action to enforce this Limited License Agreement or any of its intellectual property rights relating to thekit and/or its components.

For updated license terms, see www.qiagen.com.© 2001–2012 QIAGEN, all rights reserved.

Sample & Assay Technologies1072935 06/2012

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QuantiTect® Reverse TranscriptionHandbook

For cDNA synthesis with integrated removal of genomicDNA contaminationFor use in real-time two-step RT-PCR

March 2009


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QIAGEN Sample and Assay TechnologiesQIAGEN is the leading provider of innovative sample and assay technologies, enablingthe isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result.QIAGEN sets standards in:■ Purification of DNA, RNA, and proteins■ Nucleic acid and protein assays■ microRNA research and RNAi■ Automation of sample and assay technologiesOur mission is to enable you to achieve outstanding success and breakthroughs. Formore information, visit www.qiagen.com.

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QuantiTect Reverse Transcription Handbook 03/2009 3

ContentsKit Contents 4Shipping and Storage 4Product Warranty and Satisfaction Guarantee 4Product Use Limitations 5Technical Assistance 5Safety Information 5Quality Control 6Product Description 6Introduction 7

Principle and procedure 7Enzymatic activities of reverse transcriptase 9

Equipment and Reagents to Be Supplied by User 10Protocol■ Reverse Transcription with Elimination of Genomic DNA for Quantitative,

Real-Time PCR 11Troubleshooting Guide 14Appendix A: General Remarks on Handling RNA 18Appendix B: Preparation, Storage, Quantification, and Determination of Quality of RNA 20Appendix C: Quantitative, Real-Time Two-Step RT-PCR 23Appendix D: Recommended Controls for Quantitative, Real-Time RT-PCR 24References 25Ordering Information 26

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Kit Contents

QuantiTect Reverse Transcription Kit (10) (50) (200)Catalog no. 205310 205311 205313Number of standard reactions* 10 50 200gDNA Wipeout Buffer, 7x 100 µl 100 µl 4 x 100 µlQuantiscript® Reverse Transcriptase† 10 µl 50 µl 4 x 50 µlQuantiscript RT Buffer, 5x‡ 200 µl 200 µl 4 x 200 µlRT Primer Mix 50 µl 50 µl 4 x 50 µlRNase-Free Water 1.9 ml 1.9 ml 4 x 1.9 mlHandbook 1 1 1

* A standard reaction is 20 µl in volume with 10 pg to 1 µg total RNA.† A mixture of the QIAGEN® products Omniscript® Reverse Transcriptase and Sensiscript® Reverse

Transcriptase. Also contains RNase inhibitor.‡ Includes Mg2+ and dNTPs.

Shipping and StorageThe QuantiTect Reverse Transcription Kit is shipped on dry ice. The kit, including allreagents and buffers, should be stored immediately upon receipt at –20°C in a constant-temperature freezer.

Product Warranty and Satisfaction GuaranteeQIAGEN guarantees the performance of all products in the manner described in ourproduct literature. The purchaser must determine the suitability of the product for itsparticular use. Should any product fail to perform satisfactorily due to any reason otherthan misuse, QIAGEN will replace it free of charge or refund the purchase price. Wereserve the right to change, alter, or modify any product to enhance its performanceand design. If a QIAGEN product does not meet your expectations, simply call yourlocal Technical Service Department or distributor. We will credit your account orexchange the product — as you wish. Separate conditions apply to QIAGEN scientificinstruments, service products, and to products shipped on dry ice. Please inquire formore information.A copy of QIAGEN terms and conditions can be obtained on request, and is alsoprovided on the back of our invoices. If you have questions about product specificationsor performance, please call QIAGEN Technical Services or your local distributor (seeback cover or visit www.qiagen.com).

QuantiTect Reverse Transcription Handbook 03/20094

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Product Use LimitationsThe QuantiTect Reverse Transcription Kit is intended for research use. No claim orrepresentation is intended to provide information for the diagnosis, prevention, ortreatment of a disease.All due care and attention should be exercised in the handling of the products. Werecommend all users of QIAGEN products to adhere to the NIH guidelines that havebeen developed for recombinant DNA experiments, or to other applicable guidelines.

Technical AssistanceAt QIAGEN, we pride ourselves on the quality and availability of our technical support.Our Technical Service Departments are staffed by experienced scientists with extensivepractical and theoretical expertise in sample and assay technologies and the use ofQIAGEN products. If you have any questions or experience any difficulties regardingthe QuantiTect Reverse Transcription Kit or QIAGEN products in general, please do nothesitate to contact us.QIAGEN customers are a major source of information regarding advanced orspecialized uses of our products. This information is helpful to other scientists as well asto the researchers at QIAGEN. We therefore encourage you to contact us if you haveany suggestions about product performance or new applications and techniques.For technical assistance and more information, please see our Technical Support Center at www.qiagen.com/Support or call one of the QIAGEN Technical ServiceDepartments or local distributors (see back cover or visit www.qiagen.com).

Safety InformationWhen working with chemicals, always wear a suitable lab coat, disposable gloves,and protective goggles. For more information, please consult the appropriate materialsafety data sheets (MSDSs). These are available online in convenient and compact PDFformat at www.qiagen.com/Support/MSDS.aspx where you can find, view, and printthe MSDS for each QIAGEN kit and kit component. 24-hour emergency informationEmergency medical information in English, French, and German can be obtained 24hours a day from:Poison Information Center Mainz, GermanyTel: +49-6131-19240

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Quality ControlIn accordance with QIAGEN’s ISO-certified Quality Management System, each lot ofQuantiTect Reverse Transcription Kit is tested against predetermined specifications toensure consistent product quality.

Product Description

Component DescriptiongDNA Wipeout Buffer, 7x Buffer for effective elimination of genomic DNA

contamination from starting RNA samples.Quantiscript Reverse Developed for use in real-time two-step RT-PCR. Transcriptase Contains an optimized mixture of the QIAGEN products

Omniscript Reverse Transcriptase and Sensiscript ReverseTranscriptase, which are recombinant heterodimericenzymes expressed in E. coli. Also contains RNaseinhibitor, a 50 kDa protein that strongly inhibits RNases A,B, and C as well as human placental RNases.

Quantiscript RT Buffer, 5x Buffer optimized for reverse transcription withQuantiscript Reverse Transcriptase; contains dNTPs.

RT Primer Mix Optimized blend of oligo-dT and random primersdissolved in water. RT Primer Mix allows high cDNAyields from all regions of RNA transcripts, even from 5' regions.

RNase-Free Water Ultrapure quality, PCR-grade


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IntroductionThe QuantiTect Reverse Transcription Kit provides a fast and convenient procedure forefficient reverse transcription and effective genomic DNA elimination. The kit isdedicated for use in real-time two-step RT-PCR, and provides high cDNA yields forsensitive quantification of even low-abundance transcripts.

Principle and procedureThe QuantiTect Reverse Transcription procedure takes only 20 minutes and comprises2 main steps: elimination of genomic DNA and reverse transcription (see flowchart,next page).

Elimination of genomic DNAThe purified RNA sample is briefly incubated in gDNA Wipeout Buffer at 42°C for 2 minutes to effectively remove contaminating genomic DNA. In contrast to othermethods, the RNA sample is then used directly in reverse transcription.Accurate results in real-time RT-PCR depend on the use of primers or probes designedto eliminate or minimize detection of genomic DNA. If such primers or probes are notavailable, then genomic DNA contamination in RNA samples must be eliminated.

Reverse transcriptionAfter genomic DNA elimination, the RNA sample is ready for reverse transcription usinga master mix prepared from Quantiscript Reverse Transcriptase, Quantiscript RT Buffer,and RT Primer Mix. The entire reaction takes place at 42°C and is then inactivated at95°C. In contrast to other methods, additional steps for RNA denaturation, primerannealing, and RNase H digestion are not necessary.Quantiscript Reverse Transcriptase has a high affinity for RNA and is optimized forefficient and sensitive cDNA synthesis from 10 pg to 1 µg of RNA. This high RNAaffinity, in combination with Quantiscript RT Buffer, enables high cDNA yields, evenfrom templates with high GC-content or complex secondary structure. RT Primer Mix ensures cDNA synthesis from all regions of RNA transcripts, even from5' regions. This allows high yields of cDNA template for real-time PCR analysisregardless of where the target region is located on the transcript.

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Mix RNA,gDNA Wipeout Buffer,and RNase-free water

Incubate at 42°C for 2 min

Add Quantiscript ReverseTranscriptase, Quantiscript RTBuffer, and RT Primer Mix, and mix

Add cDNA to real-timePCR mix and distribute

Quantitative, real-time PCR

Incubate at 42°C for 15 min

Incubate at 95°C for 3 min toinactivate Quantiscript ReverseTranscriptase

QuantiTect Reverse Transcription Procedure

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Enzymatic activities of reverse transcriptaseReverse transcriptase enzymes are generally derived from RNA-containing retrovirusessuch as avian myeloblastosis virus (AMV), Moloney murine leukemia virus (MMLV), orhuman immunodeficiency virus (HIV). Quantiscript Reverse Transcriptase is from a newsource.

Figure 1. cDNA synthesis. Quantiscript Reverse Transcriptase in first-strand cDNA synthesis.

In general, reverse transcriptase is a multifunctional enzyme with 3 distinct enzymaticactivities: an RNA-dependent DNA polymerase, a hybrid-dependent exoribonuclease(RNase H), and a DNA-dependent DNA polymerase. In vivo, the combination of these3 activities allows transcription of the single-stranded RNA genome into double-stranded DNA for retroviral infection. For reverse transcription in vitro (Figure 1), thefirst 2 activities are utilized to produce single-stranded cDNA:■ RNA-dependent DNA-polymerase activity (reverse transcription) transcribes cDNA

from an RNA template. This activity of Quantiscript Reverse Transcriptase allowssynthesis of cDNA for use in quantitative, real-time PCR.

■ RNase H activity of Quantiscript Reverse Transcriptase specifically degrades onlythe RNA in RNA:DNA hybrids. Therefore, this RNase H activity affects RNAhybridized to cDNA, but has no effect on pure RNA. A separate RNA degradationstep using RNase H enzyme is not necessary prior to real-time PCR. Furthermore,the Quantiscript RNase H activity, acting during reverse transcription, mayimprove the sensitivity of subsequent real-time PCR.

mRNA

mRNA

mRNAcDNA

cDNA

AAAAAA

AAAAAA

AAAAAA

Primer annealing

Reverse transcription(RNA-dependent DNA polymerase)

RNA degradation(RNase H)

Quantitative, real-time PCR

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Equipment and Reagents to Be Supplied by UserWhen working with chemicals, always wear a suitable lab coat, disposable gloves,and protective goggles. For more information, consult the appropriate material safetydata sheets (MSDSs), available from the product supplier.

For genomic DNA elimination and reverse transcription:■ Plastic tubes (for 20 µl reactions)■ Ice■ Heating block or water bath (capable of reaching 95°C)■ Vortexer■ Microcentrifuge■ Optional: gene-specific primers

For quantitative, real-time PCR:■ Optimized kit for quantitative, real-time PCR, which includes Taq polymerase;

quantitative, real-time PCR buffer; primers; probe or SYBR® Green I dye; andnucleotides (for details, see Appendix C, page 23).

■ QIAGEN offers optimized, ready-to-run kits for highly specific and sensitive real-time PCR:■ Rotor-Gene® Kits — for ultrafast results on the Rotor-Gene Q■ QuantiFast® Kits — for fast cycling on instruments from other suppliers■ QuantiTect Kits — for standard cycling on instruments from other suppliers;

includes optional UNG pretreatmentKits are available for SYBR Green, probe, or multiplex detection. For more details,visit www.qiagen.com/PCR. For ordering information, see page 26.

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Protocol: Reverse Transcription with Elimination ofGenomic DNA for Quantitative, Real-Time PCRImportant points before starting■ The protocol is optimized for use with 10 pg to 1 µg of RNA. If using >1 µg RNA,

scale up the reaction linearly to the appropriate volume.■ Set up all reactions on ice to minimize the risk of RNA degradation.■ RNase inhibitor and dNTPs are already included in the kit components. Do not add

additional RNase inhibitor and dNTPs.■ RT Primer Mix (supplied) or gene-specific primers (not supplied) should be used.

RT Primer Mix is optimized to provide high cDNA yields for all regions of RNAtranscripts. If using gene-specific primers, we recommend using a finalconcentration of 0.7 µM or testing a range of final concentrations from 0.5 µM to 1 µM.

■ For convenience, premix RT Primer Mix and 5x Quantiscript RT Buffer in a 1:4 ratioif RT Primer Mix will be used routinely for reverse transcription. This premix isstable when stored at –20°C.

■ Separate denaturation and annealing steps are not necessary before starting thereverse-transcription reaction.

■ If using a reaction volume of 200 µl or greater for reverse transcription, make surethe reaction tube is efficiently heated (e.g., if using a heating block, carefully filleach well with a drop of water so that heat can be efficiently transferred from theblock to the tube).

■ After reverse transcription, the reaction must be inactivated by incubation at 95°Cfor 3 minutes.

■ If working with RNA for the first time, read Appendix A, page 18.■ For details on performing real-time PCR after reverse transcription, see Appendix

C, page 23. For details on appropriate controls, see Appendix D, page 24.■ Users of the FastLane® Cell cDNA Kit: If you have purchased the QuantiTect Reverse

Transcription Kit in order to perform additional reverse-transcription reactions withthe FastLane Cell cDNA Kit, follow the protocol in the FastLane Cell cDNAHandbook. Do not follow the protocol in the QuantiTect Reverse TranscriptionHandbook.

Things to do before starting■ Dissolve any precipitates in gDNA Wipeout Buffer by vortexing. If necessary,

briefly incubate the buffer at 37°C until the precipitates dissolve.

Protocol


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Procedure1. Thaw template RNA on ice. Thaw gDNA Wipeout Buffer, Quantiscript Reverse

Transcriptase, Quantiscript RT Buffer, RT Primer Mix, and RNase-free water at roomtemperature (15–25°C). Mix each solution by flicking the tubes. Centrifuge briefly to collect residual liquidfrom the sides of the tubes, and then store on ice.

2. Prepare the genomic DNA elimination reaction on ice according to Table 1. Mix and then store on ice.Note: If setting up more than one reaction, prepare a volume of master mix 10%greater than that required for the total number of reactions to be performed. Thendistribute the appropriate volume of master mix into individual tubes followed byeach RNA sample. Keep the tubes on ice.Note: The protocol is for use with 10 pg to 1 µg RNA. If using >1 µg RNA, scaleup the reaction linearly. For example, if using 2 µg RNA, double the volumes ofall reaction components for a final 28 µl reaction volume.

Table 1. Genomic DNA elimination reaction components

Component Volume/reaction Final concentrationgDNA Wipeout Buffer, 7x 2 µl 1xTemplate RNA Variable (up to 1 µg*)RNase-free water VariableTotal volume 14 µl –

* This amount corresponds to the entire amount of RNA present, including any rRNA, mRNA, viral RNA, andcarrier RNA present, and regardless of the primers used or cDNA analyzed.

3. Incubate for 2 min at 42°C. Then place immediately on ice.Note: Do not incubate at 42°C for longer than 10 min.

4. Prepare the reverse-transcription master mix on ice according to Table 2. Mix and then store on ice. The reverse-transcription master mix contains allcomponents required for first-strand cDNA synthesis except template RNA.Note: If setting up more than one reaction, prepare a volume of master mix 10%greater than that required for the total number of reactions to be performed.Note: The protocol is for use with 10 pg to 1 µg RNA. If using >1 µg RNA, scaleup the reaction linearly. For example, if using 2 µg RNA, double the volumes ofall reaction components for a final 40 µl reaction volume.

Prot

ocol


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Protocol


Table 2. Reverse-transcription reaction components

Component Volume/reaction Final concentrationReverse-transcription master mixQuantiscript Reverse Transcriptase* 1 µlQuantiscript RT Buffer, 5x†‡ 4 µl 1xRT Primer Mix‡ 1 µlTemplate RNAEntire genomic DNA 14 µl (add at step 5)elimination reaction (step 3)Total volume 20 µl –

* Also contains RNase inhibitor.† Includes Mg2+ and dNTPs.‡ For convenience, premix RT Primer Mix and 5x Quantiscript RT Buffer in a 1:4 ratio if RT Primer Mix will be

used routinely for reverse transcription. This premix is stable when stored at –20°C. Use 5 µl of the premixper 20 µl reaction.

5. Add template RNA from step 3 (14 µl) to each tube containing reverse-transcriptionmaster mix. Mix and then store on ice.

6. Incubate for 15 min at 42°C.In some rare cases (e.g., if the RT-PCR product is longer than 200 bp or if analyzingRNAs with a very high degree of secondary structure), increasing the incubationtime up to 30 min may increase cDNA yields.

7. Incubate for 3 min at 95°C to inactivate Quantiscript Reverse Transcriptase.8. Add an aliquot of each finished reverse-transcription reaction to real-time PCR mix

(see Appendix C, page 23). Store reverse-transcription reactions on ice and proceed directly with real-timePCR, or for long-term storage, store reverse-transcription reactions at –20°C. For real-time PCR, we recommend using a Rotor-Gene Kit, QuantiFast Kit, orQuantiTect Kit (see page 10).

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Troubleshooting GuideThis troubleshooting guide may be helpful in solving any problems that may arise. Formore information, see also the Frequently Asked Questions page at our TechnicalSupport Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGENTechnical Services are always happy to answer any questions you may have abouteither the information and protocol in this handbook or sample and assay technologies(for contact information, see back cover or visit www.qiagen.com).


No product, or product detected late in real-time PCR (problems occurring duringreverse transcription)a) Pipetting error or missing Check the pipets used for experimental setup.

reagent when setting up Mix all reagents well after thawing, and repeat reverse-transcription reaction the reverse-transcription reaction.

b) Incorrect setup of Be sure to set up the reaction on ice.reverse-transcription reaction

c) Volume of reverse-transcription Adding a high volume of reverse-transcriptionreaction added to the reaction to the PCR mix may reduce amplificationreal-time PCR was too high efficiency and the linearity of the reaction.

Generally, the volume of reverse-transcriptionreaction added should not exceed 10% of thefinal PCR volume.

d) Temperature of Reverse transcription should be carried out atreverse-transcription reaction 42°C. Check the temperature of your heating

block or water bath. In rare cases, whenanalyzing RNAs with a very high degree ofsecondary structure, it may be advantageous toincrease the temperature up to 50°C. However,temperatures >42°C will reduce the activity ofQuantiscript Reverse Transcriptase and thereforeaffect the cDNA yield.

e) Short incubation time The standard reverse-transcription reactionrequires a 15-min incubation. In rare cases,when analyzing RNAs with a very high degreeof secondary structure or if the RT-PCR product is longer than 200 bp, it may be advantageousto increase the incubation time to 30 min.

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f) Poor quality or incorrect Check the concentration, integrity, and purity ofamount of template RNA for the template RNA (see Appendix B, page 20)reverse-transcription reaction before starting the protocol. Mix well after

thawing the template RNA. Even minute amountsof RNases can affect synthesis of cDNA andsensitivity in RT-PCR, particularly with smallamounts of RNA.

g) RNA concentration too high Quantiscript Reverse Transcriptase is designedor too low for use with 10 pg to 1 µg RNA. If using >1 µg

RNA, scale up the reaction linearly to theappropriate volume.

h) RNA denatured Denaturation of the template RNA is notnecessary. If denaturation was performed, theintegrity of the RNA may be affected.

i) Incorrect concentration or If using a gene-specific primer for reverse degradation of primers for transcription, check the concentration andreverse-transcription reaction integrity of the primer. If necessary, perform

reverse transcription with different primerconcentrations or use the supplied RT Primer Mix.If using RT Primer Mix, be sure to use 1 µl of RTPrimer Mix in a 20 µl reaction.

j) Incubation temperature Reverse transcription should be carried out attoo high 42°C. Higher temperatures may reduce the

length of cDNA products or the activity ofQuantiscript Reverse Transcriptase. Check thetemperature of your heating block or water bath.

FastLane Cell cDNA Kit usersk) Wrong protocol followed If using the QuantiTect Reverse Transcription Kit

to perform additional reverse-transcriptionreactions with the FastLane Cell cDNA Kit, follow the protocol in the FastLane Cell cDNAHandbook.

No product, or product detected late in real-time PCR, or only primer–dimers detected (problems occurring during real-time PCR)a) PCR annealing time too short Use the annealing time specified in the protocol

for the real-time PCR kit you are using.b) PCR extension time too short Use the extension time specified in the protocol

for the real-time PCR kit you are using.

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c) Mg2+ concentration in Always start with the Mg2+ concentration PCR not optimal recommended in the protocol for the real-time

PCR kit you are using. Perform titration in 0.5 mM steps.

d) Pipetting error or missing Check the concentrations and storage conditionsreagent when setting up PCR of reagents, including primers and cDNA.

e) Taq DNA Polymerase not Ensure that the cycling program includes the hotactivated with a hot start start activation step for Taq DNA polymerase; for

details, check the instructions supplied with thepolymerase.

f) PCR product too long For optimal results, PCR products should be100–150 bp in length and should not exceed300 bp.

g) Primer design for real-time Check for the presence of PCR products by gelPCR not optimal electrophoresis or melting curve analysis. If no

specific PCR products are detected, review theprimer design.

h) Primer concentration Use the primer concentrations recommended infor real-time PCR not optimal the protocol for the real-time PCR kit you are

using.i) Insufficient number of cycles Increase the number of cycles.

j) PCR annealing temperature Decrease annealing temperature in 3°C steps.too high

k) PCR annealing temperature Increase annealing temperature in 2°C steps.too low

l) No detection activated Check that fluorescence detection was activatedin the cycling program.

m) Wrong detection step Ensure that fluorescence detection takes placeduring the extension step of the PCR cyclingprogram.

n) Real-time PCR Check for possible degradation of primers/probesprimers/probes degraded on a denaturing polyacrylamide gel.

o) Wrong dye layer/filter chosen Ensure that the appropriate layer/filter is activated.p) Insufficient starting template Increase the amount of template cDNA, if possible.q) Primer–dimers coamplified Include an additional data acquisition step in the

in real-time PCR with SYBR cycling program to avoid the detection ofGreen I primer–dimers.

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r) Detection temperature too Ensure that the detection temperature is at leasthigh in optional data 3°C lower than the Tm of the specific product.acquisition step for real-time When establishing a new primer-template PCR with SYBR Green I system, always perform a 3-step cycling reaction

first, without the optional fourth step.Multiple peaks in melting temperature analysis/multiple PCR products

Reaction set up at room To avoid nonspecific primer annealing, set up thetemperature real-time PCR in cooled reaction vessels and/or

use a Taq DNA polymerase which requires a hotstart.

High fluorescence in “No RT” control reactionsContamination with Check that the genomic DNA elimination stepgenomic DNA with gDNA Wipeout Buffer was performed

correctly: check the temperature of your heatingblock or water bath and the concentration of thereaction components.When purifying RNA, we recommend usingRNeasy® Plus Kits, which use gDNA Eliminatorcolumns or plates to remove genomic DNAcontamination (see page 29 for orderinginformation).

No linearity in ratio of CT value/crossing point to log of the template amounta) Template amount too high Do not exceed maximum recommended amounts

of template cDNA. For details, see the protocolfor the real-time PCR kit you are using.

b) Template amount too low Increase amount of template RNA, if possible.High fluorescence in “No Template” controla) Contamination of reagents Discard reaction components and repeat with

new reagents.b) Contamination during Take appropriate safety precautions (e.g., use

reaction setup filter tips).Varying fluorescence intensitya) Real-time cycler contaminated Decontaminate the real-time cycler according to

the supplier’s instructions. b) Real-time cycler no longer Recalibrate the real-time cycler according to the

calibrated supplier’s instructions.

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Appendix A: General Remarks on Handling RNAHandling RNARibonucleases (RNases) are very stable and active enzymes that generally do notrequire cofactors to function. Since RNases are difficult to inactivate and even minuteamounts are sufficient to degrade RNA, do not use any plasticware or glasswarewithout first eliminating possible RNase contamination. Although the QuantiTect ReverseTranscription Kit contains RNase inhibitor, we still recommend that care should be takento avoid inadvertently introducing RNases into the RNA sample during or after thepurification procedure. In order to create and maintain an RNase-free environment, thefollowing precautions must be taken during pretreatment and use of disposable andnondisposable vessels and solutions while working with RNA.

General handlingProper microbiological, aseptic technique should always be used when working withRNA. Hands and dust particles may carry bacteria and molds and are the most commonsources of RNase contamination. Always wear latex or vinyl gloves while handlingreagents and RNA samples to prevent RNase contamination from the surface of the skinor from dusty laboratory equipment. Change gloves frequently and keep tubes closedwhenever possible.

Disposable plasticwareThe use of sterile, disposable polypropylene tubes is recommended throughout theprocedure. These tubes are generally RNase-free and do not require pretreatment toinactivate RNases.

Nondisposable plasticwareNondisposable plasticware should be treated before use to ensure that it is RNase-free.Plasticware should be thoroughly rinsed with 0.1 M NaOH,* 1 mM EDTA* followedby RNase-free water (see “Solutions”, page 19). Alternatively, chloroform-resistantplasticware can be rinsed with chloroform* to inactivate RNases.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protectivegoggles. For more information, consult the appropriate material data sheets (MSDSs), available from theproduct supplier.

1056039_HB 19.03.2009 14:01 Uhr Seite 18


GlasswareGlassware should be treated before use to ensure that it is RNase-free. Glassware usedfor RNA work should be cleaned with a detergent,* thoroughly rinsed, and oven bakedat 240°C for 4 hours or more (overnight, if more convenient) before use. Autoclavingalone will not fully inactivate many RNases. Alternatively, glassware can be treated with DEPC* (diethyl pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water),allow to stand overnight (12 hours) at 37°C, and then autoclave or heat to 100°C for15 minutes to eliminate residual DEPC.

SolutionsSolutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is astrong, but not absolute, inhibitor of RNases. It is commonly used at a concentration of0.1% to inactivate RNases on glass or plasticware or to create RNase-free solutions andwater. DEPC inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 mlof the solution to be treated and shake vigorously to bring the DEPC into solution. Letthe solution incubate for 12 hours at 37°C. Autoclave for 15 minutes to remove anytrace of DEPC. DEPC will react with primary amines and cannot be used directly to treatTris* buffers. DEPC is highly unstable in the presence of Tris buffers and decomposesrapidly into ethanol and CO2. When preparing Tris buffers, treat water with DEPC first,and then dissolve Tris to make the appropriate buffer. Trace amounts of DEPC willmodify purine residues in RNA by carbethoxylation. Carbethoxylated RNA is translatedwith very low efficiency in cell-free systems. However, its ability to form DNA:RNA orRNA:RNA hybrids is not seriously affected unless a large fraction of the purine residueshave been modified. Residual DEPC must always be eliminated from solutions or vesselsby autoclaving or heating to 100°C for 15 minutes.Note: QIAGEN solutions, such as Quantiscript RT Buffer and RNase-free water, areguaranteed RNase-free without using DEPC treatment and are therefore free of anyDEPC contamination.


1056039_HB 19.03.2009 14:01 Uhr Seite 19


Appendix B: Preparation, Storage, Quantification, and Determination of Quality of RNAPreparation of RNAReverse transcriptases are used in vitro for first-strand cDNA synthesis with RNA as thestarting template. The efficiency of the reaction is highly dependent on the quality andquantity of the starting RNA template.It is important to have intact RNA as starting template. Even trace amounts ofcontaminating RNases in the RNA sample can cause RNA cleavage, resulting inshortened cDNA products. Chemical impurities, such as protein, poly-anions (e.g.,heparin), salts, EDTA, ethanol, and phenol, can affect the activity and processivity ofthe reverse transcriptase. To ensure reproducible and efficient reverse transcription, it isimportant to determine the quality and quantity of the starting RNA (see below).For best results, we recommend starting with RNA purified using silica-gel–membranetechnology. For ordering information, see page 29.

Storage of RNAPurified RNA may be stored at –20°C or –70°C in water. Under these conditions, nodegradation of RNA is detectable after 1 year.

Quantification of RNAThe concentration of RNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. To ensure significance, readings should begreater than 0.15. An absorbance of 1 unit at 260 nm corresponds to 44 µg of RNAper ml (A260=1 → 44 µg/ml). This relation is valid only for measurements at a neutralpH. Therefore, if it is necessary to dilute the RNA sample, this should be done in a bufferwith neutral pH. As discussed below (see “Purity of RNA”, page 21), the ratio betweenthe absorbance values at 260 and 280 nm gives an estimate of RNA purity.When measuring RNA samples, be certain that cuvettes are RNase-free, especially ifthe RNA is to be recovered after spectrophotometry. This can be accomplished bywashing cuvettes with 0.1 M NaOH,* 1 mM EDTA* followed by washing with RNase-free water (see “Solutions”, page 19). Use the buffer in which the RNA is dilutedto zero the spectrophotometer.


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An example of the calculation involved in RNA quantification is shown below:Volume of RNA sample = 100 µlDilution = 20 µl of RNA sample + 180 µl of 10 mM Tris·Cl,* pH 7.0 (1/10 dilution)Measure absorbance of diluted sample in a 0.2 ml cuvette (RNase-free):A260 = 0.2Concentration of RNA sample = 44 µg/ml x A260 x dilution factor

= 44 µg/ml x 0.2 x 10= 88 µg/ml

Total amount = concentration x volume of sample in ml= 88 µg/ml x 0.1 ml= 8.8 µg of RNA

Purity of RNAThe ratio of the readings at 260 nm and 280 nm (A260/A280) provides an estimate ofthe purity of RNA with respect to contaminants that absorb in the UV, such as protein.However, the A260/A280 ratio is influenced considerably by pH. Since water is notbuffered, the pH and the resulting A260/A280 ratio can vary greatly. Lower pH results ina lower A260/A280 ratio and reduced sensitivity to protein contamination.† For accuratevalues, we recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure RNAhas an A260/A280 ratio of 1.9–2.1‡ in 10 mM Tris·Cl, pH 7.5. Always be sure tocalibrate the spectrophotometer with the same solution.For determination of RNA concentration, however, we still recommend dilution of thesample in a buffer with neutral pH since the relationship between absorbance andconcentration (A260 reading of 1 = 44 µg/ml RNA) is based on an extinction coefficientcalculated for RNA at neutral pH (see “Quantification of RNA”, page 20).


† Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on thespectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.

‡ Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with somespectrophotometers.

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Integrity of RNAThe integrity and size distribution of total RNA can be checked by denaturing agarosegel electrophoresis and ethidium bromide staining* or by using the QIAxcel® system(www.qiagen.com/QIAxcel) or Agilent® 2100 bioanalyzer. The respective ribosomalRNAs should appear as sharp bands or peaks. The apparent ratio of 28S rRNA to 18SrRNA should be approximately 2:1. If the ribosomal bands or peaks of a specificsample are not sharp, but appear as a smear towards smaller sized RNAs, it is likelythat the sample suffered major degradation either before or during RNA purification.The Agilent 2100 bioanalyzer also provides an RNA Integrity Number (RIN) as a usefulmeasure of RNA integrity. Ideally, the RIN should be close to 10, but in many cases(particularly with tissue samples), RNA quality is greatly influenced by how well theoriginal sample was preserved.


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Appendix C: Quantitative, Real-Time Two-Step RT-PCRFor the quantification of RNA transcripts, quantitative, real-time RT-PCR is the mostsensitive and reliable method. Real-time RT-PCR begins with the reverse transcription ofRNA into cDNA, and is followed by PCR amplification of the cDNA. RNA is transcribedinto single-stranded cDNA using random primers, gene-specific primers, or oligo-dTprimers that specifically hybridize to the poly-A tail of mRNAs. The quantity of cDNA isdetermined during the exponential phase of PCR by the detection of fluorescencesignals that exceed a certain threshold. Fluorescence signals are generated byfluorophores incorporated into the PCR product (e.g., in assays using SYBR Green I dye)or by fluorophores which are coupled to short oligonucleotide probes (i.e., in probe-based assays). In real-time RT-PCR, the level of RNA transcripts is calculated from thenumber of the PCR cycle at which the threshold is exceeded. This cycle is called thethreshold cycle or the crossing point. For reliable results in quantitative, real-time PCRof cDNA generated using the QuantiTect Reverse Transcription Kit, we recommendusing a Rotor-Gene Kit, QuantiFast Kit, or QuantiTect Kit (see page 10 for moreinformation).In quantitative, real-time two-step RT-PCR, cDNA is first synthesized by reversetranscription. An aliquot of the finished reverse-transcription reaction is then used forPCR. Reverse transcription and PCR are performed sequentially in 2 separate reactiontubes. With the QuantiTect Reverse Transcription Kit, RT Primer Mix (supplied) or gene-specific primers (not supplied) can be used to synthesize cDNA for quantitative, real-time two-step RT-PCR. In addition, cDNA can be stored for later analysis. C1. Carry out reverse transcription according to the protocol on page 11, using the

QuantiTect Reverse Transcription Kit and 10 pg to 1 µg RNA.C2. Add an aliquot of each finished reverse-transcription reaction to real-time PCR mix.

Note: No more than 1/10 of the final PCR volume should derive from the finishedreverse-transcription reaction. For example, for a 50 µl PCR assay, use ≤5 µl of thefinished reverse-transcription reaction.

C3. Carry out real-time PCR as recommended by the supplier. We recommend using a Rotor-Gene Kit, QuantiFast Kit, or QuantiTect Kit (see page 10).

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Appendix D: Recommended Controls for Quantitative,Real-Time RT-PCRNo RT controlWith the QuantiTect Reverse Transcription Kit, genomic DNA is efficiently removed ina single step. However, all reverse-transcription experiments should include a negativecontrol to test for contaminating genomic DNA. Genomic DNA contamination can bedetected by performing a control reaction in which no reverse transcription is possible.This control contains all components including template RNA, except for QuantiscriptReverse Transcriptase. Reverse transcription therefore cannot take place and the onlytemplate available is contaminating genomic DNA. In rare cases in which genomicDNA is still amplified, detection of contaminating DNA can be eliminated with speciallydesigned primers or probes (Figure 2).

Primer spans an intron/exon boundary

Probe spans an intron/exon boundary

Figure 2. Primer/probe design. Primer/probe design to eliminate signals from contaminating genomic DNA.

1056039_HB 19.03.2009 14:01 Uhr Seite 24


Positive controlIn some cases, it may be necessary to include a positive control containing a knownconcentration of template. This is usually a substitute for absolute standards and is used only to test for presence or absence of the target, but does not yield detailedquantitative information. Ensure that the positive control contains at least the minimumamount of RNA required for accurate detection.

No template control (NTC)All real-time PCR quantification experiments should include an NTC containing all thecomponents of the reaction except for the template. This enables detection of carryovercontamination from previous experiments.

ReferencesQIAGEN maintains a large, up-to-date online database of scientific publicationsutilizing QIAGEN products. Comprehensive search options allow you to find the articlesyou need, either by a simple keyword search or by specifying the application, researcharea, title, etc.For a complete list of references, visit the QIAGEN Reference Database online atwww.qiagen.com/RefDB/search.asp or contact QIAGEN Technical Services or yourlocal distributor.

1056039_HB 19.03.2009 14:01 Uhr Seite 25



Product Contents Cat. no.

QuantiTect Reverse For 10 x 20 µl reactions: gDNA 205310Transcription Kit (10) Wipeout Buffer, Quantiscript Reverse

Transcriptase, Quantiscript RT Buffer, RT Primer Mix, and RNase-Free Water





AccessoriesQuantiTect Primer Assays — for use in real-time RT-PCR with SYBR Green detection (search for and order assays at www.qiagen.com/GeneGlobe)QuantiTect Primer For 200 x 50 µl reactions or VariesAssay (200)* 400 x 25 µl reactions: 10x QuantiTect

Primer Assay (lyophilized)Rotor-Gene SYBR Green PCR Kit — for ultrafast real-time PCR and two-step RT-PCR using SYBR Green I on the Rotor-Gene QRotor-Gene SYBR For 400 x 25 µl reactions: 3 x 1.7 ml 204074Green PCR Kit (400)† 2x Master Mix, 2 x 2 ml RNase-Free

WaterQuantiFast SYBR Green PCR Kit — for fast real-time PCR and two-step RT-PCR using SYBR Green IQuantiFast SYBR Green For 400 x 25 µl reactions: 3 x 1.7 ml 204054PCR Kit (400)† 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free Water

* Assays also available in 96- or 384-well plates; please inquire.† Trial-size kit and larger kit also available; please inquire.

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QuantiTect SYBR Green PCR Kit — for real-time PCR and two-step RT-PCR using SYBR Green IQuantiTect SYBR Green For 200 x 50 µl reactions: 3 x 1.7 ml 204143PCR Kit (200)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free WaterRotor-Gene Probe PCR Kit — for ultrafast real-time PCR and two-step RT-PCR using sequence-specific probes on the Rotor-Gene QRotor-Gene Probe For 400 x 25 µl reactions: 3 x 1.7 ml 204374PCR Kit (400)* 2x Master Mix, 2 x 2 ml RNase-Free

WaterQuantiFast Probe PCR Kits — for fast real-time PCR and two-step RT-PCR using sequence-specific probesFor all instruments from Applied Biosystems except the Applied Biosystems® 7500QuantiFast Probe PCR For 400 x 25 µl reactions: 3 x 1.7 ml 204254Kit (400)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free WaterFor the Applied Biosystems 7500 and instruments from other suppliersQuantiFast Probe PCR For 400 x 25 µl reactions: 3 x 1.7 ml 204354+ROX Vial Kit (400)* 2x Master Mix (without ROX dye),

210 µl ROX Dye Solution, 2 x 2 ml RNase-Free Water

QuantiTect Probe PCR Kit — for real-time PCR and two-step RT-PCR using sequence-specific probesQuantiTect Probe PCR For 200 x 50 µl reactions: 3 x 1.7 ml 204343Kit (200)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free Water Rotor-Gene Multiplex PCR Kit — for ultrafast multiplex real-time PCR and two-step RT-PCR on the Rotor-Gene QRotor-Gene Multiplex For 400 x 25 µl reactions: 3 x 1.7 ml 204774PCR Kit (400)* 2x Master Mix, 2 x 2 ml RNase-Free

Water

* Trial-size kit and larger kit also available; please inquire.

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QuantiFast Multiplex PCR Kits — for fast multiplex real-time PCR and two-step RT-PCRFor all instruments from Applied Biosystems except the Applied Biosystems 7500QuantiFast Multiplex For 400 x 25 µl reactions: 3 x 1.7 ml 204654PCR Kit (400)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free WaterFor the Applied Biosystems 7500 and instruments from other suppliersQuantiFast Multiplex For 400 x 25 µl reactions: 3 x 1.7 ml 204754PCR +R Kit (400)* 2x Master Mix (without ROX dye),

210 µl ROX Dye Solution, 2 x 2 ml RNase-Free Water

QuantiTect Multiplex PCR Kits — for multiplex real-time PCR and two-step RT-PCRFor all instruments from Applied BiosystemsQuantiTect Multiplex For 200 x 50 µl reactions: 3 x 1.7 ml 204543PCR Kit (200)* 2x Master Mix (with ROX dye),

2 x 2 ml RNase-Free WaterFor instruments from other suppliersQuantiTect Multiplex For 200 x 50 µl reactions: 3 x 1.7 ml 204743PCR NoROX Kit (200)* 2x Master Mix (without ROX dye),

2 x 2 ml RNase-Free WaterRelated products

FastLane Cell cDNA Kit — for high-speed preparation of cDNA without RNA purification for real-time RT-PCRFastLane Cell cDNA Kit (50) Buffer FCW, Buffer FCP, and 215011

components for 50 x 20 µl reverse-transcription reactions (gDNA Wipeout Buffer, Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, RT Primer Mix, and RNase-Free Water)

* Trial-size kit and larger kit also available; please inquire.

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RNeasy Plus Kits — for purification of total RNA from cells and tissues using gDNA Eliminator columns or platesRNeasy Plus Micro Kit (50) For 50 micropreps: RNeasy MinElute® 74034

Spin Columns, gDNA Eliminator Mini Spin Columns, Collection Tubes, Carrier RNA, RNase-Free Reagents and Buffers

RNeasy Plus Mini Kit (50) For 50 minipreps : RNeasy Mini Spin 74134Columns, gDNA Eliminator Mini Spin Columns, Collection Tubes, RNase-Free Reagents and Buffers

RNeasy Plus 96 Kit (12) For 12 x 96 preps: gDNA Eliminator 7419296 Plates, RNeasy 96 Plates, Elution Microtubes CL, Caps, S-Blocks, AirPore Tape Sheets, RNase-Free Water and Buffers

Rotor-Gene Kits, QuantiFast Kits, QuantiTect Kits and Assays, and FastLane Kits areintended for research use. No claim or representation is intended to provide informationfor the diagnosis, prevention, or treatment of a disease.RNeasy Plus Kits are intended for molecular biology applications. These products areneither intended for the diagnosis, prevention, or treatment of a disease, nor have theybeen validated for such use either alone or in combination with other products.Visit www.qiagen.com/geneXpression to find out more about standardized solutionsfor gene expression analysis — from RNA preparation to real-time RT-PCR

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Notes

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Trademarks: QIAGEN®, QIAxcel®, FastLane®, MinElute®, Omniscript®, QuantiFast®, Quantiscript®, QuantiTect®, RNeasy®,Sensiscript® (QIAGEN Group); Agilent® (Agilent Technologies, Inc.); Applied Biosystems® (Applera Corporation or its subsidiaries);Rotor-Gene® (Corbett Research Pty Ltd); SYBR® (Molecular Probes, Inc.). Registered names, trademarks, etc. used in this document,even when not specifically marked as such, are not to be considered unprotected by law.

Limited License Agreement

Use of this product signifies the agreement of any purchaser or user of the QuantiTect Reverse Transcription Kit to the followingterms:

1. The QuantiTect Reverse Transcription Kit may be used solely in accordance with the QuantiTect Reverse Transcription Handbookand for use with components contained in the Kit only. QIAGEN grants no license under any of its intellectual property to useor incorporate the enclosed components of this Kit with any components not included within this Kit except as described in theQuantiTect Reverse Transcription Handbook and additional protocols available at www.qiagen.com.

2. Other than expressly stated licenses, QIAGEN makes no warranty that this Kit and/or its use(s) do not infringe the rights ofthird-parties.

3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.

4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.

5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate anyacts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recov-er all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or anyof its intellectual property rights relating to the Kit and/or its components.

For updated license terms, see www.qiagen.com.

© 2005–2009 QIAGEN, all rights reserved.

1056039_HB 19.03.2009 14:01 Uhr Seite 31


www.qiagen.com

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1056039 03/2009

1056039_HB 19.03.2009 14:01 Uhr Seite 32

July 2011


QuantiFast® SYBR® Green RT-PCR Handbook

For fast, quantitative, real-time, one-step

RT-PCR using SYBR Green I

QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result.

QIAGEN sets standards in:

� Purification of DNA, RNA, and proteins

� Nucleic acid and protein assays

� microRNA research and RNAi

� Automation of sample and assay technologies

Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit www.qiagen.com.

QuantiFast SYBR Green RT-PCR Handbook 07/2011 3

Contents

Kit Contents 4

Shipping and Storage 4

Product Use Limitations 5

Product Warranty and Satisfaction Guarantee 5

Technical Assistance 5

Safety Information 6

Product Description 7

Quality Control 7

Introduction 8

One-step RT-PCR 8

Protocol

� Real-Time, One-Step RT-PCR 11

Troubleshooting Guide 14

Appendix A: Preparation, Quantification, Determination of Quality,

and Storage of RNA 18

Appendix B: Assay Design and Handling Primers 20

Appendix C: Quantifying Gene Expression Levels and Generating

Standard Curves 24

Absolute and relative quantification 24

Generating standard curves 25

Appendix D: Controls 27

Appendix E: Data Analysis 28

Appendix F: Collecting Well Factors on Bio-Rad iQ Cyclers 34

Ordering Information 36

4 QuantiFast SYBR Green RT-PCR Handbook 07/2011

Kit Contents

QuantiFast SYBR Green RT-PCR Kit (400) (2000)

Catalog no. 204154 204156

Number of reactions (25 �l/20 �l/10 �l) 400/500/ 1000

2000/2500/ 5000

2x QuantiFast SYBR Green RT-PCR Master Mix, containing: � HotStarTaq® Plus DNA Polymerase � QuantiFast SYBR Green RT-PCR Buffer � dNTP mix (dATP, dCTP, dGTP, dTTP) � ROX™ passive reference dye

3 x 1.7 ml 25 ml

QuantiFast RT Mix, a mixture of the QIAGEN products: � Omniscript® Reverse Transcriptase � Sensiscript® Reverse Transcriptase

100 �l 0.5 ml

RNase-Free Water 2 x 1.9 ml 20 ml

Handbook 1 1

Shipping and Storage The QuantiFast SYBR Green RT-PCR Kit is shipped on dry ice. The kit should be stored immediately upon receipt at –20ºC in a constant-temperature freezer and protected from light. When the kit is stored under these conditions and handled correctly, performance is guaranteed until the expiration date (see the quality-control label inside the kit box or on the kit envelope). 2x QuantiFast SYBR Green RT-PCR Master Mix can also be protected from light and stored at 2–8ºC for up to 1 month without showing any reduction in performance.

To maintain optimal performance of the QuantiFast SYBR Green RT-PCR Kit for 2000 x 25 �l reactions, we recommend storing the 25 ml master mix and the 0.5 ml RT mix as appropriately sized aliquots in sterile, polypropylene tubes.


Product Use Limitations The QuantiFast SYBR Green RT-PCR Kit is intended for molecular biology

applications. This product is not intended for the diagnosis, prevention, or

treatment of a disease.

All due care and attention should be exercised in the handling of the products.

We recommend all users of QIAGEN products to adhere to the NIH guidelines

that have been developed for recombinant DNA experiments, or to other

applicable guidelines.

Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described

in our product literature. The purchaser must determine the suitability of the

product for its particular use. Should any product fail to perform satisfactorily

due to any reason other than misuse, QIAGEN will replace it free of charge or

refund the purchase price. We reserve the right to change, alter, or modify any

product to enhance its performance and design. If a QIAGEN product does not

meet your expectations, simply call your local Technical Service Department or

distributor. We will credit your account or exchange the product — as you wish.

Separate conditions apply to QIAGEN scientific instruments, service products,

and to products shipped on dry ice. Please inquire for more information.

A copy of QIAGEN terms and conditions can be obtained on request, and is

also provided on the back of our invoices. If you have questions about product

specifications or performance, please call QIAGEN Technical Services or your

local distributor (see back cover).

Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical

support. Our Technical Service Departments are staffed by experienced

scientists with extensive practical and theoretical expertise in molecular biology

and the use of QIAGEN products. If you have any questions or experience any

difficulties regarding the QuantiFast SYBR Green RT-PCR Kit or QIAGEN

products in general, please do not hesitate to contact us.

QIAGEN customers are a major source of information regarding advanced or

specialized uses of our products. This information is helpful to other scientists as

well as to the researchers at QIAGEN. We therefore encourage you to contact

us if you have any suggestions about product performance or new applications

and techniques.


For technical assistance and more information, please see our Technical Support Center at www.qiagen.com/Support or call one of the QIAGEN Technical Service Departments or local distributors (see back cover or visit www.qiagen.com).

Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDF format at www.qiagen.com/ts/msds.asp where you can find, view, and print the MSDS for each QIAGEN kit and kit component.

24-hour emergency information

Emergency medical information in English, French, and German can be obtained 24 hours a day from:

Poison Information Center Mainz, Germany

Tel: +49-6131-19240


Product Description 2x QuantiFast SYBR Green RT-PCR Master Mix contains:

Component Description HotStarTaq Plus DNA Polymerase:

HotStarTaq Plus DNA Polymerase is a modified form of a recombinant 94 kDa DNA polymerase,

originally isolated from Thermus aquaticus. HotStarTaq Plus DNA Polymerase is provided in

an inactive state and has no enzymatic activity at ambient temperature. The enzyme is activated by

a 5-minute, 95ºC incubation step.

QuantiFast SYBR Green

RT-PCR Buffer:

Contains Tris·Cl, KCl, (NH4)2SO4, MgCl2, and

additives enabling fast cycling, including Q-Bond

QuantiFast SYBR Green RT-PCR Buffer:

Contains Tris·Cl, KCl, (NH4)2SO4, MgCl2, and additives enabling fast cycling, including Q-Bond

dNTP mix: Contains dATP, dCTP, dGTP, and dTTP of

ultrapure quality

Fluorescent dyes: SYBR Green I and ROX

QuantiFast RT Mix: Contains an optimized mixture of the QIAGEN

products Omniscript Reverse Transcriptase and

Sensiscript Reverse Transcriptase, both of which are recombinant heterodimeric enzymes

expressed in E. coli.

RNase-free water: Ultrapure quality, PCR-grade

Quality Control In accordance with QIAGEN’s ISO-certified Quality Management System, each

lot of QuantiFast SYBR Green RT-PCR Kit is tested against predetermined specifications to ensure consistent product quality.


Introduction The QuantiFast SYBR Green RT-PCR Kit provides rapid real-time quantification of RNA targets in an easy-to-handle format. The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. High specificity and sensitivity in RT-PCR are achieved by the use of the hot-start enzyme, HotStarTaq Plus DNA Polymerase, together with a specialized fast RT-PCR buffer. The buffer also contains ROX dye, which allows fluorescence normalization on certain cyclers. The optimized Omniscript and Sensiscript blend for the reverse-transcription step further enhances sensitivity. Short cycling steps without loss of PCR sensitivity and efficiency are enabled by Q-Bond®, a patent-pending additive in the RT-PCR buffer.

The kit has been optimized for use with any real-time cycler, including cyclers with standard ramping rates and cyclers with rapid ramping rates:

� Applied Biosystems: ABI PRISM® 7000, 7700, and 7900, Applied Biosystems® 7300 and 7500, GeneAmp® 5700, ViiA™ 7 Real-Time PCR System, StepOne™ and StepOnePlus™ Real-Time PCR Systems

� Bio-Rad: iCycler iQ®, iQ5, MyiQ™, DNA Engine Opticon®, DNA Engine Opticon 2, CFX96™ Real-Time PCR Detection System, CFX384™ Real-Time PCR Detection System

� Cepheid: SmartCycler®

� QIAGEN: Rotor-Gene® cyclers

� Eppendorf: Mastercycler® ep realplex

� Roche: LightCycler® 1.x, LightCycler 2.0, LightCycler 480

� Agilent (formerly Stratagene): Mx3000P®, Mx3005P®, Mx4000®

This handbook contains a general protocol for use with all these systems.

One-step RT-PCR Use of 2x QuantiFast SYBR Green RT-PCR Master Mix together with QuantiFast RT Mix allows both reverse transcription and PCR to take place in a single tube. All reagents required for both reactions are added at the beginning, so there is no need to open the tube once the reverse-transcription reaction has been started.

The components of 2x QuantiFast SYBR Green RT-PCR Master Mix include HotStarTaq Plus DNA Polymerase, QuantiFast SYBR Green RT-PCR Buffer, SYBR Green I, and ROX passive reference dye (see descriptions below). QuantiFast RT Mix contains an Omniscript and Sensiscript blend (see descriptions below).


Omniscript and Sensiscript

QuantiFast RT Mix contains an optimized Omniscript and Sensiscript blend. Both enzymes exhibit a high affinity for RNA, facilitating transcription through secondary structures that may inhibit other reverse transcriptases. Omniscript is designed for reverse transcription of RNA amounts greater than 50 ng, and Sensiscript is optimized for use with very small amounts of RNA (<50 ng). This enzyme combination provides highly efficient and sensitive reverse transcription over a wide range of RNA template amounts.

HotStarTaq Plus DNA Polymerase

HotStarTaq Plus DNA Polymerase is a modified form of QIAGEN Taq DNA Polymerase, and is provided in an inactive state and has no enzymatic activity at ambient temperature. The enzyme remains completely inactive during the reverse-transcription reaction and does not interfere with it. This prevents formation of misprimed RT-PCR products and primer–dimers during reaction setup, reverse transcription, and the first denaturation step. The enzyme is activated after the reverse-transcription step by a 5-minute, 95ºC incubation step. The hot start also inactivates the reverse-transcriptase enzymes, ensuring temporal separation of reverse transcription and PCR, and allowing both steps to be performed sequentially in a single tube. In addition, the concentration of the polymerase in the master mix is optimized to allow short extension times in the combined annealing/extension step of each PCR cycle.

QuantiFast SYBR Green RT-PCR Buffer

QuantiFast SYBR Green RT-PCR Buffer is specifically designed for fast-cycling, real-time, one-step RT-PCR using SYBR Green I. A novel additive in the buffer, Q-Bond, allows short cycling times on standard cyclers and on fast cyclers with rapid ramping rates. Q-Bond increases the affinity of Taq DNA polymerases for short single-stranded DNA, reducing the time required for primer annealing to a few seconds. This allows a combined annealing/extension step of only 30 seconds. In addition, the unique composition of the buffer supports the melting behavior of DNA, enabling short denaturation and annealing/extension times.

QuantiFast SYBR Green RT-PCR Buffer is also based on the unique QIAGEN OneStep RT-PCR buffer system. The buffer contains a balanced combination of KCl and (NH4)2SO4, which promotes a high ratio of specific to nonspecific primer binding during the annealing step of each PCR cycle. This creates stringent primer annealing conditions, leading to increased PCR specificity. When using this buffer, primer annealing is only marginally influenced by the MgCl2 concentration, so optimization by titration of Mg2+ is not required.


SYBR Green I

2x QuantiFast SYBR Green RT-PCR Master Mix contains an optimized concentration of the fluorescent dye SYBR Green I. SYBR Green I binds all double-stranded DNA molecules, emitting a fluorescent signal on binding. 2x QuantiFast SYBR Green RT-PCR Master Mix can be stored at –20ºC without loss of SYBR Green I fluorescence activity. The excitation and emission maxima of SYBR Green I are at 494 nm and 521 nm, respectively, which are compatible with use on any real-time cycler.

Passive reference dye

For certain real-time cyclers, the presence of ROX passive reference dye in real-time PCR compensates for non-PCR–related variations in fluorescence detection. Fluorescence from ROX dye does not change during the course of real-time PCR, but provides a stable baseline to which PCR-related fluorescent signals are normalized. Thus, ROX dye compensates for differences in fluorescence detection between wells due to slight variations in reaction volume or to differences in well position.

The use of ROX dye is necessary for all instruments from Applied Biosystems and is optional for the Mx3000P, Mx3005P, and Mx4000. Instruments from Bio-Rad, Cepheid, QIAGEN, Eppendorf, and Roche do not require ROX dye. The presence of ROX dye in the master mix does not interfere with real-time PCR on any instrument, since the dye is not involved in the reaction and has an emission spectrum completely different from that of SYBR Green I.


Protocol: Real-Time, One-Step RT-PCR

Important points before starting

� The QuantiFast SYBR Green RT-PCR Kit has been developed for use in a

two-step cycling protocol, with a denaturation step at 95ºC and a

combined annealing/extension step at 60ºC. This protocol will also work

for primers with a Tm well below 60ºC.

� For the highest efficiency in real-time RT-PCR using SYBR Green I, targets

should ideally be 60–200 bp in length.

� After reverse transcription, the PCR step of the RT-PCR must start with an

initial incubation step of 5 minutes at 95ºC to activate HotStarTaq Plus DNA Polymerase.

� Set up all reactions on ice to avoid premature cDNA synthesis.

� For 96-well block cyclers, we recommend a final reaction volume of 25 �l. For capillary cyclers, we recommend a final reaction volume of 20 �l. For

384-well block cyclers, we strongly recommend a final reaction volume of

10 �l.

� Always start with the Mg2+ concentration as provided in 2x QuantiFast

SYBR Green RT-PCR Master Mix.

� If using QuantiTect Primer Assays, the final concentration in the reaction

should be 1x. Also, follow the cycling protocol in Table 2.

� If using the iCycler iQ, iQ5, or MyiQ, well factors must be collected at the

beginning of each experiment. Well factors are used to compensate for any

system or pipetting nonuniformity. For details, refer to the user manual

supplied with the instrument or Appendix F (page 34).

Procedure

1. Thaw 2x QuantiFast SYBR Green RT-PCR Master Mix, template RNA,

primers, and RNase-free water. Mix the individual solutions, and

place them on ice. QuantiFast RT Mix should be taken from –20ºC

immediately before use, always kept on ice, and returned to storage

at –20ºC immediately after use.

2. Prepare a reaction mix according to Table 1.

Keep samples on ice while preparing the reaction mix.

Note: We strongly recommend starting with the Mg2+ concentration as

provided in 2x QuantiFast SYBR Green RT-PCR Master Mix.


Table 1. Reaction Setup

Volume/reaction

Component

96-well block

Capillary cycler

384-well block

Final concentration

2x QuantiFast SYBR Green RT-PCR Master Mix

12.5 �l 10 �l 5 �l 1x

Primer A* Variable Variable Variable 1 �M

Primer B* Variable Variable Variable 1 �M

QuantiFast RT Mix 0.25 �l 0.2 �l 0.1 �l

Template RNA (added at step 4)

Variable Variable Variable �100 ng/ reaction

RNase-free water Variable Variable Variable

Total reaction volume

25 �l 20 �l 10 �l

* If using QuantiTect Primer Assays, the final concentration in the reaction should be 1x.

3. Mix the reaction mix thoroughly and dispense appropriate volumes into PCR vessels or plates. Keep the PCR vessels or plates on ice.

4. Add template RNA (�100 ng/reaction) to the individual PCR vessels or wells containing the reaction mix.

5. Program your real-time cycler according to the program outlined in Table 2. Data acquisition should be performed during the combined annealing/ extension step.


Table 2. Real-Time Cycler Conditions

Step

Time

Temperature

Ramp rate

Additional comments

Reverse transcription

10 min 50ºC

PCR initial activation step

5 min 95ºC Maximal/ fast mode

HotStarTaq Plus DNA Polymerase is activated by this heating step

Two-step cycling

Denaturation 10 s 95ºC Maximal/ fast mode

Combined annealing/ extension

30 s 60ºC* Maximal/ fast mode

Perform fluorescence data collection

Number of cycles 35–40 The number of cycles depends on the amount of template RNA

* This temperature should also be used for QuantiTect Primer Assays and for all primer sets

with a Tm well below 60ºC.

6. Place the PCR vessels or plates in the real-time cycler and start the cycling program.

7. Optional: Perform melting curve analysis of the RT-PCR product(s) to verify their specificity and identity. Melting curve analysis is an analysis step built into the software of real-time cyclers. Please follow instructions provided by the supplier.

8. Optional: Check the specificity of the RT-PCR product(s) by agarose gel electrophoresis.

A step-by-step guide to software setup for your cycler can be found at www.qiagen.com/FastPCR


Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocol in this handbook or molecular biology applications (see back cover for contact information).


No product, or product detected late in RT-PCR, or only primer–dimers detected

a) PCR annealing/ extension time too short

Use the recommended annealing/extension time of 30 s.

b) Mg2+ concentration adjusted

Do not adjust the Mg2+ concentration in 2x QuantiFast SYBR Green RT-PCR Master Mix.

c) HotStarTaq Plus DNA Polymerase not activated

Ensure that the cycling program includes the HotStarTaq Plus DNA Polymerase activation step (5 min at 95ºC) as described in the protocol.

d) RT step not performed Ensure that the cycling program includes the RT step (10 min at 50ºC) as described in the protocol.

e) Pipetting error or missing reagent

Check the concentrations and storage conditions of the reagents, including primers and template nucleic acid. See Appendix B, page 20, for details on evaluating the concentration of primers. Repeat the PCR.

f) Wrong or no detection step

Ensure that fluorescence detection takes place during the combined annealing/extension step.

g) Primer concentration not optimal

Use each primer at a concentration of 1 �M, as described in the protocol. If using a 10x QuantiTect Primer Assay, the final concentration in the reaction should be 1x.

Check the concentrations of primers by spectrophotometry (see Appendix B, page 20).



h) Reaction volume too high

For 96-well block cyclers, we recommend a final reaction volume of 25 �l. For capillary cyclers, we recommend a final reaction volume of 20 �l. For 384-well block cyclers, we strongly recommend a final reaction volume of 10 �l.

i) Problems with starting template

Check the concentration, storage conditions, and quality of the starting template (see Appendix A, page 18).

If necessary, make new serial dilutions of template nucleic acid from the stock solutions. Repeat the RT-PCR using the new dilutions.

j) Insufficient amount of starting template

Increase the amount of template, if possible. Ensure that sufficient copies of the target nucleic acids are present in your sample.

k) Insufficient number of cycles

Increase the number of cycles in steps of 5 cycles.

l) RT-PCR product too long

For optimal results, RT-PCR products should be between 60 and 200 bp. RT-PCR products should not exceed 300 bp.

m) Primer design not optimal

Check for RT-PCR products by melting curve analysis (see Appendix E, page 28) or gel electrophoresis. If no specific RT-PCR products are detected, review the primer design guidelines (see Appendix B, page 20). Alternatively, use QuantiTect Primer Assays, which are predesigned primer sets for real-time RT-PCR (see Ordering Information, page 36).

n) No detection activated Check that fluorescence detection was activated in the cycling program.

o) Primers degraded Check for possible degradation of primers on a denaturing polyacrylamide gel.



Applied Biosystems, Bio-Rad, QIAGEN, and Agilent systems only:

p) Wrong detection

channel/filter chosen

Ensure that the correct detection channel is

activated or the correct filter set is chosen for

SYBR Green I.

LightCycler systems only:

q) Chosen fluorescence

gains too low

When using software versions earlier than 3.5,

ensure fluorescence gain for channel 1 is set to

“15”.

Primer–dimers and/or nonspecific RT-PCR products

a) Mg2+

concentration

adjusted

Do not adjust the Mg2+

concentration in 2x

QuantiFast SYBR Green RT-PCR Master Mix.

b) Primer design not

optimal

Check for RT-PCR products by melting curve

analysis (see Appendix E, page 28) or gel

electrophoresis. If no specific RT-PCR products

are detected, review the primer design guidelines

(see Appendix B, page 20). Alternatively, use

QuantiTect Primer Assays, which are

predesigned primer sets for real-time RT-PCR

(see Ordering Information, page 36).

c) RT-PCR product too

long

For optimal results, RT-PCR products should be

between 60 and 200 bp. RT-PCR products

should not exceed 300 bp.

d) Primers degraded Check for possible degradation of primers on a

denaturing polyacrylamide gel.

e) Contamination of RNA

sample with genomic

DNA

Design primers that span exon–exon boundaries,

so that only cDNA targets can be amplified and

detected. Alternatively, use QuantiTect Primer

Assays, which are predesigned primer sets that

avoid amplification of genomic DNA where

possible (see Ordering Information, page 36).

Alternatively, treat the RNA sample with DNase

to digest the contaminating genomic DNA.



Applied Biosystems, Bio-Rad, and Agilent systems:

f) Wavy curve at high

template amounts for

highly expressed

targets

In the analysis settings, reduce the number of

cycles used for background calculation (if your

real-time cycler allows you to do so) or reduce

the amount of template.

LightCycler systems only:

g) RT-PCR mix not in

capillary tip

Centrifuge the capillary to bring the RT-PCR mix

into the capillary tip.

h) Capillary not pushed

down completely

Ensure that the capillary is completely pushed

down in the LightCycler carousel.

i) Wrong detection

channel

Make sure that the correct channel is chosen.


Appendix A: Preparation, Quantification, Determination of Quality, and Storage of RNA

Template preparation and quality

Since PCR consists of multiple rounds of enzymatic reactions, it is more sensitive to impurities such as proteins, phenol/chloroform, salts, and EDTA than single-step enzyme-catalyzed reactions. Purity of nucleic acid templates is particularly important for real-time PCR, since contaminants can interfere with fluorescence detection. QIAGEN offers a complete range of RNA purification systems, ensuring the highest-quality templates for real-time RT-PCR, including RNeasy® Kits for preparation of RNA from various sources, and Oligotex® Kits (low-throughput) and TurboCapture Kits (high-throughput) for mRNA purification. QIAGEN also offers a range of BioRobot® systems for automated purification of RNA. For more information about these products, visit www.qiagen.com.

Determining concentration and purity of RNA

The concentration of RNA should be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. For accuracy, absorbance readings at 260 nm should fall between 0.15 and 1.0. An absorbance reading of 1.0 at 260 nm in a 1 cm detection path corresponds to an RNA concentration of 40 �g/ml.

Note that absorbance measurements cannot discriminate between DNA and RNA. Depending on the method used for template preparation, RNA may be contaminated with DNA, and this will result in misleadingly high A260 values. It is particularly important to bear this in mind when preparing standards for absolute quantification (see Appendix C, page 24).

The ratio between the absorbance values at 260 nm and 280 nm gives an estimate of the purity of RNA. To determine RNA purity, we recommend measuring absorbance in 10 mM Tris·Cl,* pH 7.5. Pure RNA has a A260/A280 ratio of 1.9–2.1.† Lower ratios indicate the presence of contaminants such as proteins.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and

protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs), available from the product supplier.

† Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some spectrophotometers.


Storage of RNA

Purified RNA should be stored at –20ºC or –70ºC in RNase-free water. When RNA is purified using QIAGEN kits, no degradation is detectable for at least 1 year under these conditions. Diluted solutions of nucleic acids (e.g., dilution series used as standards) should be stored in aliquots and thawed once only. We recommend storage of aliquots in siliconized tubes where possible. This avoids adsorption of nucleic acids to the tube walls, which would reduce the concentration of nucleic acids in solution.


Appendix B: Assay Design and Handling Primers Important factors for successful quantitative, real-time RT-PCR include the design of optimal primer pairs, the use of appropriate primer concentrations, and the correct storage of primers.

Assay design

For guaranteed results in gene expression analysis experiments, we recommend using QuantiTect Primer Assays (see Ordering Information, page 36). If designing your own primers, please follow the guidelines provided in Table 3. Since fluorescence from SYBR Green I increases strongly upon binding of the dye to any double-stranded DNA, it is particularly important to minimize nonspecific primer annealing by careful primer design.

Table 3. General Guidelines for Design of Primers

Length 18–30 nucleotides

GC content 40–60%

Tm For best results, use commercially available oligo-design software such as OLIGO 6 (oligo.net) or Web-based tools such as Primer3 (frodo.wi.mit.edu/cgi-bin/primer3/ primer3_www.cgi)* to determine primer Tms.

Simplified formula for estimating melting temperature (Tm):

Tm = 2ºC x (number of [A+T]) + 4ºC x (number of [G+C])

Whenever possible, design primer pairs with similar Tm values.

Sequence � Always check the specificity of primers by performing a BLAST® search (www.ncbi.nlm.nih.gov/blast). Ensure that primer sequences are unique for your template sequence.

� Ensure the length of the PCR product is less than 200 bp.

Table continued on next page

* Rozen, S. and Skaletsky, H.J. (2000) Primer3 on the WWW for general users and for

biologist programmers. In: Krawetz, S. and Misener, S., eds. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Totowa, NJ: Humana Press, pp. 365–386.


Table 3. Continued

Sequence � Avoid complementarity of 2 or more bases at the 3' ends of primer pairs to minimize primer–dimer formation.

� Avoid mismatches between the 3' end of primers and the template sequence.

� Avoid runs of 3 or more Gs or Cs at the 3' end.

� Avoid a 3'-end T. Primers with a T at the 3' end have a greater tolerance of mismatch.

� Avoid complementary sequences within a primer sequence and between the primer pair.

� Commercially available computer software (e.g., OLIGO 6) or Web-based tools (e.g., Primer3) can be used for primer design. Use the software to minimize the likelihood of formation of stable primer–dimers.

Special consider-ations for design of RT-PCR primers and probes

� Design primers so that one half hybridizes to the 3' end of one exon and the other half to the 5' end of the adjacent exon (see Figure 1). The primers will therefore anneal to cDNA synthesized from spliced mRNAs, but not to genomic DNA, eliminating detection of contaminating DNA.

� Alternatively, RT-PCR primers should be designed to flank a region that contains at least one intron. Products amplified from cDNA (no introns) will be smaller than those amplified from genomic DNA (containing introns). If possible, select a target with very long introns: the RNA target may then be preferentially amplified because of the higher PCR efficiency of this shorter PCR product without introns. If genomic DNA is detected (i.e., presence of amplification product in “No RT” control), treat the template RNA with RNase-free DNase. Alternatively, redesign primers to avoid amplification of genomic DNA.


Figure 1. Primer design. Primer design to A eliminate or B detect amplification from

contaminating genomic DNA.

Handling and storing primers

Guidelines for handling and storing primers are provided in Table 4 below. For

optimal results, we recommend only combining primers of comparable quality.

Table 4. General Guidelines for Handling and Storing Primers

Storage buffer

Lyophilized primers should be dissolved in a small volume

of low-salt buffer to give a concentrated stock solution

(e.g., 100 �M). We recommend using TE (10 mM Tris·Cl,

1 mM EDTA, pH 8.0) for standard primers.

Storage Primers should be stored in TE in small aliquots at –20ºC.

Standard primers are stable under these conditions for at

least 1 year. Repeated freeze–thaw cycles should be

avoided, since they may lead to degradation.

Table continued on next page


Table 4. Continued

Dissolving primers

Before opening a tube containing lyophilized primer, spin the tube briefly to collect all material at the bottom of the tube. To dissolve the primer, add the required volume of TE, mix, and leave for 20 minutes to allow the primer to completely dissolve. Mix again and determine the concentration by spectrophotometry as described below.

We do not recommend dissolving primers in water. They are less stable in water and some may not dissolve easily.

Concentration Spectrophotometric conversion for primers:

1 A260 unit = 20–30 �g/ml

To check primer concentration, the molar extinction coefficient (H260) can be used:

A260 = H260 x molar concentration of primer

If the H260 value is not given on the data sheet supplied with the primers, it can be calculated from the primer sequence using the following formula:

H260 = 0.89 x [(A x 15,480) + (C x 7340) + (G x 11,760) + (T x 8850)]

Example Concentration of diluted primer: 1 �M = 1 x 10–6 M

Primer length: 24 nucleotides with 6 each of A, C, G, and T bases

Calculation of expected A260: 0.89 x [(6 x 15,480) + (6 x 7340) + (6 x 11,760) + (6 x 8850)] x (1 x 10–6) = 0.232

The measured A260 should be within +/– 30% of the theoretical value. If the measured A260 is very different to the theoretical value, we recommend recalculating the concentration of the primers, or having the primers resynthesized.

Primer quality The quality of 18–30mers can be checked on a 15% denaturing polyacrylamide gel; a single band should be seen. Please contact QIAGEN Technical Services or your local distributor (see back cover) for a protocol.


Appendix C: Quantifying Gene Expression Levels and Generating Standard Curves This appendix provides information on quantification of target nucleic acids. Further information can be found in Critical Factors for Successful Real-Time PCR. To obtain a copy, contact QIAGEN Technical Services, or visit www.qiagen.com/literature/defaultbrochures.aspx to download a PDF.

Absolute and relative quantification Target nucleic acids can be quantified using either absolute quantification or relative quantification. Absolute quantification determines the absolute amount of a target (expressed as a copy number or concentration), whereas relative quantification determines the ratio between the amount of a target and the amount of a reference nucleic acid, usually a suitable housekeeping gene. This normalized value can then be used to compare, for example, differential gene expression in different samples.

Absolute quantification

The absolute amount of a target nucleic acid is determined using external standards. The sequence of the standards is usually the same as or very similar to the target sequence, but the primer binding sites of the standards must be identical to those in the target sequence. This ensures that both the standards and the target are amplified with equivalent efficiencies, which is essential for absolute quantification. A standard curve (plot of CT value/crossing point against log of amount of standard) is generated using different dilutions of the standard. The target and each of the standards are amplified in separate tubes. The CT value of the target is compared with the standard curve, allowing calculation of the initial amount of the target. It is important to select an appropriate standard (see page 25).

Relative quantification

With this method, the amounts of the target genes and the reference gene within the same sample are determined, and ratios are calculated between each target gene and the reference gene. These normalized values can then be used to compare, for example, differential gene expression in different samples. The most common application of this method is analysis of gene expression or, more generally, determination of the abundance of RNA targets. The expression level of the reference gene, such as a housekeeping gene, must not vary under different experimental conditions, or in different states of the same tissue (e.g., “disease” versus “normal” samples). The level is therefore used as a reference value for quantification. The quantification procedure differs depending on whether the target genes and the reference gene are amplified


with comparable or different efficiencies. For determination of PCR efficiency, see below.

Different amplification efficiencies

The amplification efficiencies of target and reference genes are sometimes different due to differences in primer binding sites, PCR product sequences, and PCR product sizes. If this is the case, we recommend generating several standard curves (see below), one for each target or reference gene. The standards can be, for example, total RNA prepared from a reference cell line. The amounts of the target genes and the reference gene are determined by comparing their CT values with the corresponding standard curve. Ratios can then be calculated between each target gene and the reference gene. Since the expression of the reference gene remains the same between different samples, the ratio of the target genes to the reference gene will vary depending on the expression of the target genes (e.g., in different tissues).

Comparable amplification efficiencies

If the amplification efficiencies of the target and reference genes are the same, only the standard curve for the reference gene needs to be generated. The amounts of the target and reference genes are determined by comparing their CT values with this standard curve.

Alternatively, the comparative or ''CT method can be used. This involves comparing CT values, and does not require preparation of standard curves. This method can only be used if the amplification efficiencies of the target and reference genes are nearly equivalent.

Determination of PCR efficiency

To compare the amplification efficiencies of, for example, 2 target genes (targets A and B), prepare different dilutions each target. Amplify the different dilutions by real-time RT-PCR. Subtract the CT values of target A from the CT values of target B. Plot the differences in CT values against the logarithm of amount of target. If the slope of the resulting straight line is <0.1, the amplification efficiencies are comparable.

Generating standard curves Standard curves can be used in both absolute and relative quantification. To generate a standard curve, at least 5 different amounts of the standard should be quantified, and the amount of unknown target should fall within the range of the standard curve. Reactions should be carried out in at least triplicate, especially when quantifying standards of low copy number.


Standards

For absolute quantification of RNA molecules (see page 24), the copy number

or concentration of the nucleic acids used as standards must be known. In

addition, standards should show the following features:

� Primer binding sites identical to the target to be quantified

� Sequence between primer binding sites identical or highly similar to target

sequence

� Sequences upstream and downstream from the amplified sequence

identical or similar to “natural” target

For quantification of RNA, we strongly recommend using RNA molecules as

standards. Depending on the sequence and structure of the target and the

efficiency of reverse transcription, only a proportion of the target RNA will be

reverse transcribed. The DNA generated during reverse transcription serves as

the template for amplification in the subsequent PCR. The use of RNA standards

takes the variable efficiency of the RT reaction into account.

RNA standards can be created by cloning part or all of the transcript of interest

into a standard cloning vector. The insert can be generated by RT-PCR from

total RNA or mRNA, or by PCR from cDNA. The cloning vector must contain an

RNA polymerase promoter such as T7, SP6, or T3. Ensure that in vitro

transcription of the insert leads to generation of the sense transcript. After in

vitro transcription, plasmid DNA must be removed completely with RNase-free

DNase, since residual plasmid DNA will lead to errors in spectrophotometric

determination of RNA concentration and will also serve as a template in the

subsequent PCR. Furthermore, ensure that the RNA used as a standard does not

contain any degradation products or aberrant transcripts by checking that it

migrates as a single band in gel electrophoresis.

After determination of RNA concentration by spectrophotometry, the copy

number of standard RNA molecules can be calculated using the following

formula:

(X g/�l RNA / [transcript length in nucleotides x 340]) x 6.022 x 1023 = Y

molecules/�l

Example

Transcript length: 500 nucleotides

Concentration: 30 ng/�l = 30 x 10–9 g/�l

Calculation: (30 x 10–9 g/�l / [500 x 340]) x 6.022 x 1023 = 1.1 x 1011

molecules/�l

An alternative to the use of in vitro transcripts as RNA standards is the use of a

defined RNA preparation (e.g., from a cell line or virus preparation), for which

the absolute concentration of the target has already been determined.


Appendix D: Controls

No template control (NTC)

All quantification experiments should include an NTC, containing all the components of the reaction except for the template. This enables detection of contamination.

RT control

All RT-PCR experiments should include a negative control to test for contaminating DNA. However, detection of this contamination can be eliminated by using suitable primers (see Table 3, pages 20). If it is not possible to use such primers, DNA contamination can be detected by performing a control reaction in which no reverse transcription is possible. The control RT reaction contains all components including template RNA, except for the reverse transcriptase enzyme. Reverse transcription therefore cannot take place. When an aliquot of this control is used as a template in PCR, the only template available is contaminating DNA.

Positive control

In some cases it may be necessary to include a positive control, containing a known concentration of template. This is usually a substitute for absolute standards and is used to test only for presence or absence of the target, but does not yield detailed quantitative information. Ensure that the positive control contains at least the minimum amount of DNA required for accurate detection.


Appendix E: Data Analysis When carrying out data analysis, follow the recommendations provided by the manufacturer of your real-time cycler. Fundamental guidelines for data analysis and some important considerations are given below.

General considerations for data analysis

Real-time PCR data are produced as sigmoidal-shaped amplification plots (when using a linear scale), in which fluorescence is plotted against the number of cycles (Figure 2, page 29).

� The threshold cycle (CT value) serves as a tool for calculation of the starting template amount in each sample. This is the cycle in which there is the first detectable significant increase in fluorescence.

� The optimal threshold setting depends on the reaction chemistries used for PCR. Therefore, an optimal threshold setting established for another kit may not be suitable for the QuantiFast SYBR Green RT-PCR Kit, and may need to be adjusted.

� The method for determination of CT values differs depending on the real-time cycler used. Check the handbook or the software help file for your real-time cycler for details on threshold settings.

� Whenever possible, select the option for automatic calculation of threshold and baseline for your real-time cycler. However, note that the default values for data analysis in the cycler software will not always provide the most accurate results.

� Most real-time cyclers contain a function that determines the noise level in early cycles, where there is no detectable increase in fluorescence due to PCR products (usually referred to as the baseline settings). Adjust the settings for this function.


Figure 2. Typical amplification plot. Amplification plots showing increases in fluorescence from 2 samples (A and B). Sample A contains a higher amount of starting template than sample B.

Applied Biosystems instruments

Before performing data analysis on Applied Biosystems instruments, read the important points below. For further details, refer to the handbook, supplementary literature, or software help file for the instrument being used.

ABI PRISM 7900

The following points only apply to SDS software version 2.1 or higher. If you work with a version earlier than 2.1, we recommend updating your SDS software to the most recent version.

� The analysis settings for the baseline and threshold need to be set.

� Details on data analysis are available in the SDS 2.1 online help (from the “Help” menu, select “SDS Online Help” and enter a search term).

� If you observe wells with a CT value that strongly deviates from those of replicate wells when using the automatic analysis settings, you should record the positions of these unusual wells and reanalyze the plate.

� If you observe problems using the automatic calculation option, you may configure the analysis settings manually. Note that the default


values for the analysis settings entered in the software will not always provide the most accurate results.

ABI PRISM 7700

The following points only apply to SDS software version 1.7 or higher. If

you work with a version earlier than 1.7, we recommend updating your

SDS software to the most recent version.

� Check baseline and threshold settings.

� Analyze the plate. You may wish to save this setup in a separate file.

� Export the CT values for the assays if you want to perform data

analysis (e.g., using a spreadsheet program).

� Note that the default values for the analysis settings entered in the software will not always provide the most accurate results.

ABI PRISM 7000

The following points only apply to software version 1.1 or higher. If you

work with a version earlier than 1.1, we recommend updating your SDS

software to the most recent version.


� Details on data analysis are provided in the online help (from the

“Help” menu, select “Contents and Index” and enter a search term).

� If you observe wells with a CT value that strongly deviates from those

of replicate wells, you should record the positions of these unusual

wells and reanalyze the plate.

� If you observe problems using the automatic option, you may

configure the analysis settings manually. Note that the default values for the analysis settings entered in the software will not always provide the most accurate results.


Applied Biosystems 7300

The following points only apply to software version 1.22 or higher. If you work with a version earlier than 1.22, we recommend updating your SDS software to the most recent version.


� Details on data analysis are provided in the online help (from the “Help” menu, select “Contents and Index” and enter a search term).

� If you observe wells with a CT value that strongly deviates from those of replicate wells, you should record the positions of these unusual wells and reanalyze the plate.

� If you observe problems using the automatic option, you may configure the analysis settings manually. Note that the default values for the analysis settings entered in the software will not always provide the most accurate results.

Applied Biosystems 7500

The following points only apply to software version 1.22 or higher. If you work with a version earlier than 1.22, we recommend updating your SDS software to the most recent version.


� If you observe wells with a CT value that strongly deviates from those of replicate wells, you should record the positions of these unusual wells and reanalyze the plate.

� If you observe problems using the automatic option, you may configure the analysis settings manually. Note that the default values for the analysis settings entered in the software will not always provide the most accurate results.

� Details on data analysis are provided in the online help (from the “Help” menu, select “Contents and Index” and enter a search term).


LightCycler system

There are 2 different methods of calculating crossing points: the fit point and

the second derivative maximum method.

Fit point method: The principle of this method is the same as that used for

the Applied Biosystems instruments. Use the arithmetic

mode of baseline adjustment when analyzing data

obtained with SYBR Green I.

Noise band: The noise band must be set according to the

threshold in the log-linear phase of PCR.

Fit points: These are a defined number of reading points

in the log-linear phase, used for calculation of a straight

line that represents the linear portion of the amplification

plot. The number of fit points can be changed by the user.

Crossing point: This is the cycle at which the straight line

(calculated using fit points) crosses the noise band.

Second derivative maximum method:

The point at which the maximal increase of fluorescence

within the log-linear phase takes place is calculated by

determining the second derivative maxima of the

amplification curves. The software calculates at which

cycle number this point is reached. It is not necessary to

set a noise band.

Standard curves

Standard samples with known template amounts are defined in the

“sample setup” view. The results from all wells defined as standards are

used following the run for the generation of a standard curve. The CTs or

crossing points are plotted against the log of the template amount,

resulting in a straight line. CT values for these samples and the standard

curve are then used to calculate the amount of starting template in

experimental samples.

Experiment report

The experiment report is a summary of the PCR results. At the end of

experiments, sample names, template amounts, CT values or crossing

points, and standard deviations are listed.


Melting curves

All cyclers can perform a melting curve (except for the ABI PRISM 7700 with sequence detection software earlier than 1.7).

To carry out melting curve analysis, the temperature is increased very slowly from a low temperature (e.g., 65ºC) to a high temperature (e.g., 95ºC). At low temperatures, all PCR products are double stranded, so SYBR Green I binds to them and fluorescence is high, whereas at high temperatures, PCR products are denatured, resulting in rapid decreases in fluorescence.

The fluorescence is measured continuously as the temperature is increased and plotted against temperature. A curve is produced, because fluorescence decreases slightly through the lower end of the temperature range, but decreases much more rapidly at higher temperatures as the melting temperatures of nonspecific and specific PCR products are reached. The detection systems calculate the first derivatives of the curves, resulting in curves with peaks at the respective Tms. Curves with peaks at a Tm lower than that of the specific PCR product indicate the formation of primer–dimers, while diverse peaks with different Tms or plateaus indicate production of nonspecific products or a smear.

Figure 3. Melting curve analysis. Melting curve analysis of 2 samples (A and B). Sample A yields only 1 peak resulting from the specific amplification product (primer–dimers not coamplified). Sample B shows a peak from the specific product and a peak at a lower temperature from amplification of primer–dimers.


Appendix F: Collecting Well Factors on Bio-Rad iQ Cyclers Bio-Rad® iQ cyclers (e.g., iCycler iQ, iQ5, and MyiQ) need to collect well factors at the start of each real-time PCR experiment to compensate for any excitation or pipetting nonuniformity. When performing SYBR Green based real-time PCR, dynamic well factors cannot be collected from the experimental plate unless the PCR master mix has been spiked with fluorescein, an additional fluorophore. This is because SYBR Green fluoresces insufficiently in the initial PCR step, where there is insufficient double-stranded DNA to bind SYBR Green and allow fluorescence. Alternatively, external well factors can be collected from an external well factor plate containing only fluorescein solution. In our experience, collecting external well factors is a more reliable and convenient alternative to collecting dynamic well factors when using QuantiFast SYBR Green Kits on Bio-Rad cyclers.

If using a QuantiFast SYBR Green Kit on the iCycler iQ system, follow the procedure below to prepare and run an external well factor plate.

Procedure

F1. Dilute 10x External Well Factor Solution (Bio-Rad, cat. no. 170-8794; contains fluorescein) to a 1x concentration with distilled water.

F2. Distribute the diluted solution into the wells of a PCR plate and seal with optically clear sealing film.

The volume of diluted solution per well depends on the real-time PCR volume. For example, if the PCR volume will be 25 �l, then distribute 25 �l of diluted solution per well.

F3. Briefly centrifuge the external well factor plate, place it into the Bio-Rad iQ cycler, and close the lid.

F4. Select the SYBR Green thermal protocol and plate setup files, and click “Run with selected Protocol”.

F5. In the “RunPrep” screen, select External Plate as “Well Factor” and click “Begin Run”.

The iCycler iQ system automatically inserts a 3-cycle protocol, External.tmo in front of your thermal protocol to collect optical data.


F6. After well factors are calculated, the Bio-Rad iQ cycler pauses. Replace the external well factor plate with your experimental plate. Click “Continue Running Protocol” to start your experiment. Note: Once the external well factor plate is prepared, it can be reused

several times (over 250 times) until the iCycler iQ system indicates that the

fluorophore intensity is insufficient to calculate well factors. Store the

external well factor plate at –20ºC between experiments, and thaw and

centrifuge it before use. Be sure to protect the plate from exposure to light

when not in use.


Ordering Information Product Contents Cat. no.

QuantiFast SYBR Green RT-PCR Kit (400)

For 400 x 25 �l reactions: 3 x 1.7 ml 2x Master Mix (contains ROX dye), 100 �l RT Mix, 2 x 1.9 ml RNase-Free Water

204154

QuantiFast SYBR Green RT-PCR Kit (2000)

For 2000 x 25 �l reactions: 25 ml 2x Master Mix (contains ROX dye), 0.5 ml RT Mix, 20 ml RNase-Free Water

204156

Accessories

QuantiTect Primer Assays — for use in real-time RT-PCR with SYBR Green detection (search for and order assays at www.qiagen.com/GeneGlobe)

QuantiTect Primer Assay (200)

For 200 x 50 �l reactions or 400 x 25 �l reactions: 10x QuantiTect Primer Assay (lyophilized)

Varies

Related products

QuantiFast SYBR Green PCR Kit — for fast, quantitative, real-time PCR and two-step RT-PCR using SYBR Green I

QuantiFast SYBR Green PCR Kit (80)

For 80 x 25 �l reactions: 1 ml 2x Master Mix (contains ROX dye), 1.9 ml RNase-Free Water

204052


For 400 x 25 �l reactions: 3 x 1.7 ml 2x Master Mix (contains ROX dye), 2 x 1.9 ml RNase-Free Water

204054


For 2000 x 25 �l reactions: 25 ml 2x Master Mix (contains ROX dye), 20 ml RNase-Free Water

204056

QuantiFast Probe RT-PCR Kits — for fast, quantitative, real-time, one-step RT-PCR using sequence-specific probes

For all instruments from Applied Biosystems except the Applied Biosystems 7500:

QuantiFast Probe RT-PCR Kit (400)

For 400 x 25 �l reactions: 3 x 1.7 ml 2x Master Mix (contains ROX dye), 100 �l RT Mix, 2 x 1.9 ml RNase-Free Water

204454



QuantiFast Probe RT-PCR Kit (2000)

For 2000 x 25 �l reactions: 25 ml 2x Master Mix (contains ROX dye), 0.5 ml RT Mix, 20 ml RNase-Free Water

204456

For the Applied Biosystems 7500 and instruments from Bio-Rad, Cepheid, QIAGEN, Eppendorf, Roche, and Agilent:

QuantiFast Probe RT-PCR +ROX Vial Kit (400)

For 400 x 25 �l reactions: 3 x 1.7 ml 2x Master Mix (without ROX dye), 210 �l ROX Dye Solution, 100 �l RT Mix, 1.9 ml RNase-Free Water

204554

QuantiFast Probe RT-PCR +ROX Vial Kit (2000)

For 2000 x 25 �l reactions: 25 ml 2x Master Mix (without ROX dye), 1.05 ml ROX Dye Solution, 0.5 ml RT Mix, 20 ml RNase-Free Water

204556

QuantiFast Probe PCR Kits — for fast, quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes

For all instruments from Applied Biosystems except the Applied Biosystems 7500:

QuantiFast Probe PCR Kit (80)

For 80 x 25 �l reactions: 1 ml 2x Master Mix (contains ROX dye), 1.9 ml RNase-Free Water

204252


For 400 x 25 �l reactions: 3 x 1.7 ml 2x Master Mix (contains ROX dye), 2 x 1.9 ml RNase-Free Water

204254


For 2000 x 25 �l reactions: 25 ml 2x Master Mix (contains ROX dye), 20 ml RNase-Free Water

204256

For the Applied Biosystems 7500 and instruments from Bio-Rad, Cepheid, QIAGEN, Eppendorf, Roche, and Agilent:

QuantiFast Probe PCR +ROX Vial Kit (80)

For 80 x 25 �l reactions: 1 ml 2x Master Mix (without ROX dye), 45 �l ROX Dye Solution, 1.9 ml RNase-Free Water

204352




For 400 x 25 �l reactions: 3 x 1.7 ml 2x Master Mix (without ROX dye), 210 �l ROX Dye Solution, 2 x 1.9 ml RNase-Free Water

204354


For 2000 x 25 �l reactions: 25 ml 2x Master Mix (without ROX dye), 1.05 ml ROX Dye Solution, 20 ml RNase-Free Water

204356

QuantiTect Reverse Transcription Kit — for fast cDNA synthesis for sensitive real-time two-step RT-PCR

QuantiTect Reverse Transcription Kit (50)

For 50 x 20 �l reactions: gDNA Wipeout Buffer, Quantiscript® Reverse Transcriptase, Quantiscript RT Buffer, RT Primer Mix, and RNase-Free Water

205311

QuantiTect Reverse Transcription Kit (200)

For 200 x 20 �l reactions: gDNA Wipeout Buffer, Quantiscript Reverse Transcriptase, Quantiscript RT Buffer, RT Primer Mix, and RNase-Free Water

205313

RNeasy Mini Kit — for purification of total RNA from animal cells, animal tissues, and yeast, and for RNA cleanup

RNeasy Mini Kit (50)* 50 RNeasy Mini Spin Columns, Collection Tubes (1.5 ml and 2 ml), RNase-Free Reagents and Buffers

74104

RNeasy Plus Mini Kit — for purification of total RNA from animal cells and tissues using gDNA Eliminator columns

RNeasy Plus Mini Kit (50)

50 RNeasy Mini Spin Columns, 50 gDNA Eliminator Mini Spin Columns, Collection Tubes, RNase-Free Reagents and Buffers

74134

Oligotex Direct mRNA Mini Kit — for purification of poly A+ mRNA directly from animal cells or tissues

Oligotex Direct mRNA Mini Kit (12)*

For 12 mRNA minipreps: 420 �l Oligotex Suspension, Small Spin Columns, Collection Tubes (1.5 ml), RNase-Free Reagents and Buffers

72022

* Other kit sizes and formats available; please inquire.



TurboCapture 96 mRNA Kit — for rapid and easy mRNA purification from cultured cells in 96-well format

TurboCapture 96

mRNA Kit (1)*

1 x TurboCapture 96 mRNA Plate, and

RNase-Free Buffers

72250

RNeasy Protect Bacteria Mini Kit — for in vivo stabilization of the gene expression profile in bacteria and subsequent RNA purification

RNeasy Protect Bacteria

Mini Kit (50)†

RNeasy Mini Kit (50) and RNAprotect®

Bacteria Reagent (2 x 100 ml)

74524

For up-to-date licensing information and product-specific disclaimers, see the

respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and

user manuals are available at www.qiagen.com or can be requested from

QIAGEN Technical Services or your local distributor.

Visit www.qiagen.com/geneXpression to find out more about standardized solutions for gene expression analysis — from RNA preparation to real-time RT-PCR

* Other kit sizes and formats available; please inquire.

† Other kit format available; please inquire.


Notes


Notes


Notes

Trademarks: QIAGEN®, BioRobot®, HotStarTaq®, Oligotex®, Omniscript®, Q-Bond®, QuantiFast®, Quantiscript®, QuantiTect®, RNAprotect®, RNeasy®, Rotor-Gene®, Sensiscript® (QIAGEN Group); Mx3000P®, Mx3005P®, Mx4000® (Agilent Technologies); SmartCycler® (Cepheid); CFX96™, CFX384™, DNA Engine Opticon®, iCycler iQ®, MyiQ™ (Bio-Rad Laboratories, Inc.); Mastercycler® (Eppendorf AG); ABI PRISM®, Applied Biosystems®, GeneAmp®, ROX™, StepOne™, StepOnePlus™, SYBR®, ViiA™ (Life Technologies Corporation); BLAST® (National Library of Medicine); LightCycler® (Roche Group). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.

Oligotex Kits are not available in Japan.

Use of this product (QuantiFast SYBR Green RT-PCR Kit) is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. The purchase of this product includes a limited, nontransferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser's own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

The purchase of this product (QuantiFast SYBR Green RT-PCR Kit) includes a limited, non-transferable license under U.S. Patent No. 5,871,908 and all continuations and divisionals, and corresponding claims in patents and patent applications outside the United States, owned by Roche Diagnostics GmbH, for internal research use for non-in vitro diagnostics applications with authorized reagents with regard to Melting Curve Analysis. No right is conveyed, expressly, by implication or estoppel, under any other patent or patent claims owned by Roche Diagnostics GmbH, or by any other Party.

NOTICE TO PURCHASER: LIMITED LICENSE The purchase of this product (QuantiFast SYBR Green RT-PCR Kit) includes a limited, non-transferable right to use the purchased amount of the product to perform Applied Biosystem’s patented Passive Reference Method for the purchaser's own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. For information on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

© 2007–2011 QIAGEN, all rights reserved.

1068906 07/2011 Sample & Assay Technologies

www.qiagen.com

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