Instruction of Use Kit

7/29/2019 Instruction of Use Kit

1/21www.roche-applied-science.com

MagNA Pure 96 Cellular RNALarge Volume Kit

y Version 07Content version: June 2012

Prefilled reagents for the isolation of cellular RNA from up to 1 106 culturedcells, or up to 800 l whole blood, or whole blood stabilized in PAXgene BloodRNA tubes, or up to 25 mg fresh- frozen tissue, or tissue stabilized in RNAlater,or from 1 10 m sections from formalin-fixed, paraffin-embedded tissue, usingthe MagNA Pure 96 Instrument.

Cat. No. 05 467 535 001 Kit for 3 96 isolations

Store the kit at 15 to 25C Keep the kit away from magnets. When properly stored, kit

components are stable until theexpiration date printed on the label.

For life science research only.Not for use in diagnostic procedures.


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Table of Contents

2 www.roche-applied-science.com

1. What this Product Does ................................................................................................. 3Number of Tests 3

Kit Contents 3

Storage and Stability 3

Additional Equipment and Reagents Required 4

Application 5Assay Time 5

2. How to Use this Product ............................................................................................... 62.1 Before You Begin 6

Precautions 6

Purification Protocols 8

Sample Material 10

Reagents 11

Preparation of Working Solutions 11

Controls 11

2.2 Pre-Isolation Steps 12

Cultured Cells 12

PAXgene Tubes 12

Whole blood 12

Tissue homogenization for fresh-frozen tissue and tissue stabilized in RNAlater 12

Deparaffinization of FFPE tissue sections 13

2.3 Isolation Procedure 13

General Remarks 13

Procedure 14

Storage of RNA Eluates 14

3. Results ............................................................................................................................ 14

Experimental Results 144. Troubleshooting ............................................................................................................ 15

5. Additional Information on this Product ................................................................... 17How this Product Works 17

Test Principle 17

Quality Control 17

6. Supplementary Information ........................................................................................ 186.1 Conventions 18

Text Conventions 18

Symbols 18

Abbreviations 186.2 Changes to Previous Version 18

6.3 Ordering Information 19

6.4 Trademarks 20

6.5 Disclaimer of License 20

6.6 Regulatory Disclaimer 20


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What this Product Does

www.roche-applied-science.com 3

MagNA Pure 96 Cellular RNA Large Volume Kit y Version 07

1. What this Product Does

Number of Tests 3 96 isolations from up to 1 106 cultured cells. 3 96 isolations of 400 l whole blood or a half PAXgene tube. 3 48 isolations of 800 l whole blood or a whole PAXgene tube. 3 48 isolations from up to 25 mg fresh-frozen tissue.

3 96 isolations from up to 20 mg fresh-frozen tissue or tissue stabilized inRNAlater.

3 96 isolations from 110 m sections from formalin-fixed,paraffin-embedded tissue.

The kit is designed to process up to 288 samples in a maximum of twelveruns. For details, see section Isolation Protocols.

Kit Contents

Storage andStability

Kit components are stable at +15 to +25C until the expiration date printedon the label.

The kit is shipped at ambient temperature.

Component Label Content

Tray 1 Reagent Tray 1 3 trays

Container 1 Wash Buffer I 1 container for removing impurities

Container 2 Wash Buffer II 1 container for removing impurities

Tray 2 Reagent Tray 2 3 trays

Container 1 Lysis/BindingBuffer

1 container for cell lysis and binding of RNA

Container 2 Proteinase K 1 container for digestion of proteins

Container 3 Proteinase K

Incubation Buffer

1 container

for digestion of proteins

Container 4 DNase IncubationBuffer

1 container for digestion of DNA

Container 5 Elution Buffer 1 container for elution of RNA

Bottle 1 Magnetic GlassParticles

6 bottles MGP suspension for binding RNA

Bottle 2 DNase 6 glass vials

lyophilizate for digestion of DNA

Bottle 3 DNase IncubationBuffer

3 bottles for reconstitution of DNase


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Reagents, including reconstituted DNase, can be stored unsealed for32hours at +15 to +25C on the stage of the instrument.

Store reconstituted DNase at +2 to +8C for up to 28 days. Seal the cap ofthe bottle with parafilm after each use on the MagNA Pure 96 Instrument.Remove parafilm again when DNase is reused on the instrument.

Parafilm is not needed for sealing the caps of MGP bottles. One set of reagent trays (tray 1 and tray 2) can be used for up to four individ-

ual runs with the MagNA Pure 96 Instrument. Once opened, reagent trayscan be used for additional runs on the same MagNA Pure 96 Instrumentwithin 28 days after proper sealing, with sealing foil, and stored at +15 to+25C.

It is only possible to reuse partially used reagent cartridges on the sameMagNA Pure 96 Instrument. The MagNA Pure 96 Software for each instru-ment tracks inventory using reagent barcodes, and recognizes partiallyused reagents and tip trays, handling them appropriately in the next run.

When reagent trays are not properly sealed, or stored for longer than

28days, evaporation may negatively affect performance. When storing output plates outside the MagNA Pure 96 Instrument, or lon-ger than 32 hours on the instrument stage, seal the plate with a sealing foil.

AdditionalEquipment andReagentsRequired

Additional equipment and reagents required when using the MagNA Pure 96Cellular RNA Large Volume Kit with the MagNA Pure 96 Instrument: MagNA Pure 96 System Fluid (Internal) or (External)* MagNA Pure 96 Sealing Foil* Standard laboratory equipment Pipettes and nuclease-free, aerosol-preventive tips to predispense samples

into the MagNA Pure 96 Processing Cartridge: Standard length tips, or

optional, extra-long tips of 10 cm length. Centrifuge tubes (e.g., for PAXgene Blood RNA tubes) Phosphate buffered saline (PBS) Optional, RNA/DNA Stabilization Reagent for Blood/Bone Marrow* Optional, PAXgene Blood RNA tubes (Cat. No. 762165, available from

PreAnalytiX) Optional, new lids for PAXgene Blood RNA tubes (e.g., flexible Vacucap

Closures (16 mm), available from VWR) Optional, MagNA Pure LC RNA Tissue Lysis Buffer/Refill* (for fresh-frozen

tissue applications) Optional, MagNA Lyser Instrument* or similar device for homogenization of

fresh-frozen tissue Optional, MagNA Lyser Green Beads* Optional, MagNA Pure DNA Tissue Lysis Buffer (for FFPE tissue applications) Optional, Proteinase K (solution)* for FFPE tissue applications Vortex mixer or multiple vortex mixer Optional, Xylol, for FFPE tissue applications Optional, Ethanol, for FFPE tissue applications RNAlater

* available from Roche Applied Science


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Application The MagNA Pure 96 Cellular RNA Large Volume Kit is specifically designed toisolate highly purified cellular RNA from cultured cells, or whole blood stabi-lized with the RNA/DNA Stabilization Reagent for Blood/Bone Marrow, orwhole blood stored in PAXgene Blood RNA tubes, or from fresh-frozen tissue,or tissue stabilized in RNAlater, or from 1 10 m sections from forma-lin-fixed, paraffin-embedded tissue, using the MagNA Pure 96 Instrument.

Purified RNA can be used for RT-PCR with LightCycler Instruments or stan-dard thermal block cyclers.

Assay Time MagNA Pure 96 Instrument setup requires approximately 5 to 10 min. Totaltime for automated isolation of cellular RNA from 96 samples is approximately75 min (85 min for isolation of cellular RNA from blood prepared in PAXgenetubes), and 90 min for the isolation of cellular RNA from whole blood samples.

Additional hands-on time may be required for manual pre-isolation steps(e.g., preparation of cultured cells), depending on the protocol. Fordetailed information, see section Pre-Isolation Steps.


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How to Use this Product



2. How to Use this Product

2.1 Before You Begin

Precautions I) Handling Requirements

Perform sample preparation, RT-PCR setup, and the RT-PCR run in separatelocations.

Do not use a kit after its expiration date has passed.

Wear disposable gloves and change them frequently.

Some buffers contain dangerous or hazardous compounds. For detailedinformation, see Figure 1 (reagent tray 1), Figure 2 (reagent tray 2), and thefollowing table. Do not allow these reagents to touch the skin, eyes, ormucous membranes. If contact does occur, wash the affected area immedi-ately with large amounts of water. If the reagents are spilled, dilute the spillwith water before wiping it up.

Do not allow reagents containing guanidine thiocyanate to contact sodiumhypochlorite (bleach) solution or acids. These mixtures produce a highly toxicgas. This precaution is particularly important to be aware of when cleaningthe MagNA Pure 96 Waste Cover.

Fig. 1: Reagent Tray 1 Fig. 2: Reagent Tray 2

Component Label Dangerous/Hazardous Compounds

Tray 1 Reagent Tray 1

Container 1 Wash Buffer I Guanidine hydrochloride Ethanol

Container 2 Wash Buffer II Ethanol

Tray 2 Reagent Tray 2


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II) Laboratory Procedures

Handle all samples as if potentially infectious. Use safe laboratory proce-dures. As the sensitivity and titer of potential pathogens in sample materialcan vary, the operator must optimize pathogen inactivation. Follow theappropriate measures according to local safety regulations.

Do not eat, drink, or smoke in the laboratory area.

Do not pipette by mouth.

Wear protective disposable gloves, laboratory coats, and eye protectionwhen handling samples and kit reagents.

Wash hands thoroughly after handling samples and reagents.

III) Waste Handling

Material Safety Data Sheets (MSDS) are available on the Roche AppliedScience homepage (www.roche-applied-science.com), or upon request fromthe local Roche office.

Discard unused reagents and waste in accordance with country, federalstate, and local regulations.

Wear protective disposable gloves, laboratory coats, and eye protection whenhandling samples and kit reagents.

To discard reagents from the containers, follow the procedure below:

Container 1 Lysis/BindingBuffer

Guanidine thiocyanate

Container 2 Proteinase K Proteinase K

Container 3 Proteinase KIncubation Buffer

Urea

Container 4 DNase IncubationBuffer

Container 5 Elution Buffer

Bottle 1 Magnetic GlassParticles

Isopropanol

Bottle 2 DNase DNase

Bottle 3 DNase IncubationBuffer

Pierce the foil in the corner of one container in the reagent tray with asolid plastic disposable (e.g., a cell culture pipette).

Fold back the foil and discard the liquid in the specific container forwaste.

Discard the contents of all containers by repeating steps 1 and 2 untilall containers are empty.

Component Label Dangerous/Hazardous Compounds


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PurificationProtocols

To perform RNA isolations with the MagNA Pure 96 Cellular RNA Large Vol-ume Kit, pre-installed purification protocols are available. The elution volumemust be chosen using the software menu.

Regularly check for new versions of the purification protocols.For downloading protocols that are currently not installed, follow the proce-

dure below:

For additional details, contact your local Roche representative.

Different protocols are optimized for a specific sample material.Do not use a protocol for a sample material other than specified below.Doing so will negatively affect the performance of the isolation process andmay lead to clumping and loss of MGPs, as well as cross-contamination ofsamples, or even damage to the instrument.

Download the protocol (.zip file format) fromwww.magnapure96.com/ (see Download System Protocols), andsave the file onto a USB stick.

Log on to the MagNA Pure 96 Software as admin user.

Insert the USB stick with the protocol into the corresponding drive.

Unzip the.zip file.

In the MagNA Pure 96 Software, select the Utilities tab.

In the Utilities tab, select the Tools sub-tab. ClickImport Protocol.

The Import Protocoldialog box opens.

In the Import Protocoldialog box, select the respective purificationprotocol saved on the USB stick [Files of Type Protocols (.tdf)].

ClickOpen.The selected purification protocol is imported.

Exit the MagNA Pure 96 Software and restart the control unit.

Restarting the control unit is a prerequisite for using newlyinstalled purification protocols.

Protocol Name Sample Material Elution Volume1)

Cellular RNA LV Up to 1 106 culturedcells resuspended in200 l PBS

50, 100, or 200 l

RNA PAXgene LV PAXgene pellet resus-pended in 400 l PBS

100 or 200 l

RNA PAXgene HalfTube LV

half PAXgene pellet con-tained in 200 l PBS

50 or 100 l


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Use the most up-to-date protocol version available for purification of RNA. Only specified types of sample material can be combined in the same run. One set of reagent trays (tray 1 and tray 2) can be used for up to four indi-

vidual runs with the same MagNA Pure 96 Instrument. Pierced reagenttrays can be stored for 28 days after the first use.To prevent evaporation and contamination of reagents after use, thereagent trays have to be sealed with a sealing foil. Up to three sealing foilscan be used to cover the reagent trays.

1) The concentration of cellular RNA in the eluate, and the sensitivity indownstream applications can be increased by choosing a lower elution

volume. This may however reduce the elution efficiency, and overall RNAyield compared to using higher elution volumes. To increase total yield, werecommend elution volumes of 100 or 200 l.

2) For some tissue samples more than the specified amount of homoge-nized tissue can be used (e.g., brain).

* When using the RNA Blood LV 800 protocol, the sample volume isdivided to produce two samples and is processed in two separate wells ofthe MagNA Pure 96 Processing Cartridge. If less samples than multiples of8 are processed using the RNA Blood LV 800 protocol, it is required to

RNA Blood LV 400 400 l whole blood stabi-lized with 500 l RNA/DNA StabilizationReagent for Blood/Bone

Marrow

50, 100, or 200 l

RNA Blood LV 800* 800 l whole blood stabi-lized with 1000 l RNA/DNA StabilizationReagent for Blood/BoneMarrow

100 or 200 l

RNA Tissue FFStandard LV

up to 10 mg homogenizedtissue from fresh-frozenor RNAlater stabilized tis-sue samples in a volume

of 350 l2)

50, 100, or 200 l

RNA Tissue FFHigh LV**

up to 25 mg homogenizedtissue from fresh-frozentissue samples in a vol-ume of 700 l2)

100 or 200 l

RNA Tissue FFPE LV Deparaffinized anddigested FFPE tissuesections in a volume of150 l

50 or 100 l

Protocol Name Sample Material Elution Volume1)


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specify dummy sample data in the sample table and fill the correspondingpositions in the processing cartridge with 1800 l PBS until the next multi-ple of 8 is reached.

** When using the RNA Tissue FF High LV protocol, the sample volume isdivided to produce two samples and is processed in two separate wells ofthe MagNA Pure 96 Processing Cartridge.

Sample Material For optimal results in downstream procedures, particulary in real-time RT-PCRdo not process samples with higher volume or cell count than the selectedpurification protocol is designed to handle. Doing so can affect the perfor-mance of the isolation process, may lead to clumping and loss of MGPs, aswell as cross-contamination of samples, or even damage to the instrument.

Treat all samples as potentially infectious.

I) Cultured Cells

Cell pellets can be stored at 15 to25C for several weeks. Cultured cells resuspended in 200 l PBS.

Never use more sample material than this kit is designed to handle (e.g.,use not more than 1 106 cells). Doing so will negatively affect the perfor-mance of the isolation process.

II) PAXgene Blood RNA Tubes

Pellet of a whole PAXgene tube suspended in 400 l PBS (or optionally, onlyhalf of that suspension). The PAXgene Blood RNA tube is designed to hold 2.5ml whole blood.

III) Whole blood

The stabilized whole blood (see section 2.2 below) can be stored for 2 days

at +15 to +25C or for 1 month at - 80C.IV) Fresh-frozen tissue

Up to 25 mg fresh-frozen tissue samples (e.g., liver, kidney, lung, muscle, tail ofmammalian species) can be used after homogenization.

For some tissue samples, so-called "easy-to-lyse" tissues, more than thespecified amount of homogenized tissue can be used (e.g., brain).

When purifying 25 mg fresh-frozen tissue samples with high amounts ofnucleic acids (e.g. spleen), an additional DNase digestion step may berequired.

V) Tissues stabilized in RNAlater5 mg of RNAlater stabilized tissue can be used after homogenization.

VI) FFPE tissue

1 10 m sections from formalin-fixed, paraffin-embedded tissue.

Please be aware that section thickness as well as yield and quality of theisolated RNA are strongly related to type of tissue, age of sample as wellas fixation protocol used.


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Reagents Kit components, except DNase, are ready-to-use. Ensure that kit components are equilibrated to +15 to +25C before use. If

reagents are used at temperatures outside this recommended range, thekit may not function properly.

Preparation of

WorkingSolutions

Before starting, prepare the working solutions as described below:

Reconstitute DNase just before first time use.

Controls For control reactions, prepare the following:

Positive control, using a sample material positive for the target.

Negative control, using a sample material negative for the target.

Extraction control, using phosphate buffered saline (PBS) in place of asample.

Reagent Preparation/Comments Storage

DNase For each reagent tray set (tray 1 and tray2, for 96 samples), reconstitute two glassbottles of DNase (bottle 2) with 3 ml fromone bottle of DNase incubation buffer(bottle 3). Close the bottles and mix wellby inverting. Do not vortex. Dissolved, aclear to slightly opaque solution isobtained. Transfer all liquid to the original

plastic bottle labeled DNase incubationbuffer (bottle 3), and place a check in thebox DNase added on the label. Closethe bottle with the original lid. Mix byinverting the bottles five times.

Once reconstituted,the DNase is stablefor 28 days at +2 to+8C.


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2.2 Pre-Isolation Steps

Cultured Cells For cultured cells grown in suspension, gently spin down cells (e.g., for 5 minat 300 g). Wash the cell pellet using PBS. Cell pellets can be stored at 15to 25C for several weeks. Remove culture media (or PBS) completely.Resuspend cells in cold phosphate buffered saline (PBS) by pipetting or shak-ing the tube until the cell pellet is resuspended. Monolayer cultured cellsshould be collected using trypsinization, prior to the procedure describedabove. The required sample volume is 200 l. Ensure there are not more than1 106 cells/200 l.

PAXgene Tubes For collection, storage, and transportation of whole blood in PAXgene BloodRNA tubes, follow the PAXgene instructions. The PAXgene Blood RNA tube isdesigned to hold 2.5 ml whole blood.

After blood sample collection, centrifuge PAXgene Blood RNA tubes in aswing-out rotor with round bottomed tube adapters at 3,000 to 5,000 g for10 min. Decant the supernatent as completely as possible. A reddish to

brownish pellet will be clearly visible. Add 400 l PBS to the PAXgene BloodRNA tube pellet. Close the tube using the PAXgene Blood RNA tube lid. Donot mix up lids from different samples, decant only one PAXgene Blood RNAtube at a time. Alternatively, use a new lid.

Vortex the pellet until it is resuspended completely. When using a multitubevortexer, vortex full speed for multiples of 30 sec until the pellet is resus-pended.

When purifying RNA using the RNA PAXgene LV protocol, transfer thewhole volume (400 l) of the resuspended pellet from one PAXgene BloodRNA tube into one well of a MagNA Pure 96 Processing Cartridge.

When purifying RNA using the RNA PAXgene Half Tube LV protocol, trans-fer 200 l of the pellet resuspended in 400 l from one PAXgene Blood RNAtube into one well of a MagNA Pure 96 Processing Cartridge.

Maintain PAXgene Blood RNA tubes for at least 2 hours at +15 to +25Cbefore starting RNA isolation.

Do not use the content from more than one PAXgene Blood RNA tube perisolation to avoid clumping during the purification run.

Whole blood Stabilize 400 l (or 800 l) whole blood with 500 l (or 1000 l) RNA/DNAStabilization Reagent for Blood/Bone Marrow*, and mix thoroughly.

Tissue

homogenizationfor fresh-frozentissue and tissuestabilized inRNAlater

MagNA Lyser treatment: Transfer up to 10 mg (or up to 25 mg) tissue sample

into a MagNA Lyser Green Beads Tube containing 400 l (or 800 l)MagNA Pure LC RNA Tissue Lysis Buffer.

For some tissue samples, so-called "easy-to-lyse" tissues, more than thespecified amount of homogenized tissue can be used (e.g., brain): Up to20 mg homogenized tissue for the RNA Tissue FF Standard LV and up to50 mg for the RNA Tissue FF High LV protocol. This requires validation bythe user.

Avoid cooling at this step. Homogenize the tissue in the MagNA Lyser Instru-ment for 30 to 50 seconds (depending on the tissue type).


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If necessary, chill on ice for 90 s and repeat the procedure. To minimize activity of present nucleases, perform homogenization as fast

as possible. When homogenizing more than two samples, RIN valuesobtained using the Agilent Instrument may be lower due to prolongedtime at +15 to +25C. Tissue thawing can produce RNA degradation byRNases.

Deparaffinizationof FFPE tissuesections

To one 5 10 m section (1 cm 1 cm) in a 1.5 ml reaction tube add 800 lXylol, incubate for 5 min and mix by overhead shaking.

Add 400 l absolute ethanol and mix. Centrifuge 2 min at maximum speedand discard supernatant.

Add 1 ml absolute ethanol and mix by overhead shaking. Centrifuge for2min at maximum speed and discard supernatant.

Invert tube and blot briefly on a paper towel to get rid of residual ethanol. Drythe tissue pellet for 10 min at +55C.

Add 110 l MagNA Pure DNA Tissue Lysis Buffer.

Add 50 l Proteinase K solution and mix. Then incubate at +55C until com-plete dissolution of the tissue. The required sample volume is 150 l.

2.3 Isolation Procedure

General Remarks The following procedures are used to process 96 samples at the same time.When other sample numbers than multiples of 8 (Software version 1.1 and 2.0)or 24 (Software version 1.0) are used, the instrument will process the emptypositions until the next multiple of 8 or 24 is reached. For a detailed descriptionof the instrument setup and handling, refer to the MagNA Pure 96 SystemQuick Guide and Customer Training Guide (software version 2.0).

Ensure that kit components are equilibrated to +15 to +25C before use.

When the reagents are used at temperatures outside this recommendedrange, the purification may not function properly.

Ensure that all containers are inserted correctly into the reagent trays,prior to placing them on the stage.

Ensure that instructions are followed for type and amount of sample mate-rial (see section Sample Material). Using inappropriate types andamounts of sample material may cause clumping, which may lead to lowyield and purity of cellular RNA, as well as cross-contamination and inhibi-tion of downstream assays (e.g., RT-PCR).

After the run has finished, carefully inspect the instrument for any signs of

spillage. If spillage has occurred, clean the instrument as described in theMagNA Pure 96 System Operators Guide.

Clean the waste cover after each run, as described in theMagNA Pure 96 System Operators Guide. Do not use sodium hypochlo-rite (bleach) solution or acids for the first cleaning step, because this mayproduce highly toxic gas in combination with reagents containing guani-dine thiocyanate.


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Results



Procedure For a detailed description on how to prepare and perform a purification run,refer to the MagNA Pure 96 System Quick Guide and Customer TrainingGuide (Software version 2.0).

Storage of RNAEluates

To ensure stability of the eluted RNA, proceed immediately with RT-PCRsetup. Do not store the eluted RNA on the MagNA Pure 96 Stage for a lon-ger period of time; up to 32 hours stability on the cooled sample rack posi-tion 4 are possible.

For storage, close the output plate with the MagNA Pure 96 Sealing Foil andstore at 15 to 25C or60 to 80C. Store the RNA in aliquots if neces-sary, so that purified RNA is not repeatedly frozen and thawed.

For long-term storage, we recommend transferring eluates to an archiveplate.

After thawing eluates, mix gently by pipetting up and down ten timesbefore performing any downstream steps, e.g., RT-PCR or OD measure-ments. Mixing volumes should be at least half of the eluate volume.

Results may not be reproducible when RNA is not premixed and distrib-uted homogenously before pipetting.

3. Results

ExperimentalResults

High quality results with the MagNA Pure 96 Cellular RNA Large Volume Kitwere demonstrated using HeLa, K562 cultured cells, whole blood, PAXgenetubes, fresh-frozen tissues, tissue stabilized in RNAlater, and FFPE tissue sec-tions. High yield, purity, and integrity of cellular RNA were obtained using

spectrophotometry, electrophoresis and RT-PCR. Independent from the extraction method, RNA extracted from FFPE sec-

tions is often degraded due to formalin fixation.


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Troubleshooting



4. Troubleshooting

Possible Cause Recommendation

Clumping of

beads orpresence ofbeads in the out-put plate

Too much or inappropri-

ate sample material orinefficient homogenization.

Reduce sample material to values recom-

mended in the section Sample Material. Use only the specified types of sample material

(see section Sample Material).

MGPs were magnetizedprior to use.

Avoid contact between MGPs and magnetsprior to use.

Store kit appropriately.

RNA is degraded Improper storage ofsamples.

Use fresh-frozen samples, or tissue samplesstabilized in RNAlater.

Avoid the use of samples that have beenstored at ambient temperature.

Nuclease contamination Avoid contamination of disposables andreagents with nucleases.

Tissue homogenizationwas not fast enough.

Perform homogenization for fresh-frozen tis-sues as rapidly as possible to avoid RNase deg-radation. If necessary, chill on ice for 90 s andrepeat the procedure.

Poor or no RNAyield

Sample did not containenough cells.

Count cells before use. For optimal number ofcells, refer to section Sample Material.

Storage of samples wasnot optimal.

Use fresh-frozen samples, or tissue samplesstabilized in RNAlater.

Avoid the use of samples that have beenstored at ambient temperature.

Too much or wrongsample material.

Reduce sample material to values recom-mended in section Sample Material.

Use only specified types of sample material(see section Sample Material).

Poor RNA purity Too many cells in thesample

Reduce number of cells to the values recom-mended in section Sample Material by dilut-ing the sample.

Drops of sample material

on the walls of the wells

Pipette samples to the bottom of each well of

the processing cartridge.Poor RT-PCRperformance

Poor purity of RNA Too much sample material used for isolation.Adjust input material to the values recom-mended in section Sample Material.

Avoid sample material on the walls of wellswhen pipetting samples into the processingcartridge.


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Troubleshooting



Black particles inthe output plate

Magnetic glass particlesare present in the outputplate.

Low numbers of magnetic glass particles donot affect PCR or RT-PCR assays on theLightCycler Instrument or conventional ther-mal block cycler.

RT-PCR reagents and pro-tocols were not optimal.

Verify reagents and protocols using a positiveand negative control.

Eluates show aslight color

Drops of sample materialon the walls of the wells.

Pipette samples to bottom of wells of the pro-cessing cartridge.

Use the automated sample transfer function topipette samples into the wells of another pro-cessing cartridge. This ensures transfer with-out contaminating the walls of wells withsample material.

Too much or inappropri-

ate sample material.

Use only the specified types of sample material

(see section Sample Material).Sediments in thetarget plate(PAXgene proto-cols only)

Overloading due to highblood cell content

Centrifuge the target plate (e.g., for 2 min at1,500 g). Alternatively, use the RNA PAXgeneHalf Tube LV protocol.

These sediments do not affect RT-PCRassays.

Possible Cause Recommendation


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Additional Information on this Product



5. Additional Information on this Product

How this ProductWorks

The MagNA Pure 96 Cellular RNA Large Volume Kit is used together with theMagNA Pure 96 Instrument to purify high-quality, intact RNA from up to288 samples. Isolated RNA can be eluted in 50, 100, or 200 l (depending on

the Elution Volume selected in the MagNA Pure 96 Software). Purified RNAmeets the quality standards required for sensitive and quantitative RT-PCRusing the LightCycler Instruments.

Test Principle RNA isolation uses MagNA Pure Magnetic Glass Particle Technology. The keysteps of a MagNA Pure 96 RNA isolation are:

Quality Control The kit is function tested using the following procedures:Cellular RNA is isolated from K562 cells. High quality purified RNA is verifiedusing agarose gel electrophoresis, OD260/280, and RT-PCR using theLightCycler 480 Instrument.

Sample material is lysed, nucleic acids are released, and nucleasesare denatured.

Nucleic acids bind to the silica surface of the MGPs, due to the chao-

tropic salt conditions and high ionic strength of the lysis/binding buf-fer.

Genomic DNA is removed by incubation with DNase.

MGPs with bound RNA are magnetically separated from the rest ofthe lysed sample.

Unbound substances (e.g., proteins, cell debris, PCR inhibitors) areremoved by washing steps.

Purified RNA is eluted from the MGPs.


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Supplementary Information



6. Supplementary Information

6.1 Conventions

Text Conventions To make information consistent and easy to understand, the following textconventions are used throughout this document:

Symbols In this document, the following symbols are used to highlight important infor-mation:

Abbreviations In this document, the following abbreviations are used:

6.2 Changes to Previous Version

Editorial changes

New splitted protocol for the isolation of RNA from up to 25 mg fresh-frozentissue samples.

Text Convention Usage

Numbered stageslabeled, etc.

Stages in a process that usually occur in the orderlisted.

Numbered instructionslabeled, etc.

Steps in a procedure that must be performed inthe order listed.

Symbol Description

Information Note:Additional information about the current topic or procedure.

Important Note:Information critical to the success of the procedure or use of theproduct.

Abbreviation MeaningMGPs Magnetic glass particles

PBS Phosphate buffered saline

RT-PCR Reverse transcription polymerase chain reaction

SW Software


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6.3 Ordering Information

Roche Applied Science offers a large selection of reagents and systems for lifescience research. For a complete overview of related products and guides, visitand bookmark our homepage: www.roche-applied-science.com and our Spe-cial Interest Sites including:

Automated Sample Preparation (MagNA Lyser Instrument, MagNA PureCompact System, MagNA Pure LC System, and MagNA Pure 96 System):www.magnapure.com and www.magnapure96.com.

Real-time PCR Systems (LightCycler Carousel-Based System,LightCycler 480 System, LightCycler 1536 System, RealTime ready qPCRassays, and Universal ProbeLibrary): www.lightcycler.com.

Product Pack Size Cat. No.

Instruments MagNA Pure 96 Instrument 1 instrument, controlunit and accessories

05 195 322 001

MagNA Lyser Instrument 1 instrument (110 V.)1 instrument (220 V.)plus 2 MagNA LyserRotors, a MagNA LyserRotor Cooling Block,and a MagNA LyserRotor Stand

03 358 968 00103 358 976 001

Reagents and Kits MagNA Pure 96 DNA and Viral NASmall Volume Kit

3 192 purifications 05 467 497 001

MagNA Pure 96 DNA and Viral NALarge Volume Kit

3 96 purifications 05 467 454 001

RNA/DNA Stabilization Reagentfor Blood/Bone Marrow

1 Bottle 11 934 317 001

MagNA Pure 96 System Fluid(Internal)

2 Containers 05 467 098 001

MagNA Pure 96 System Fluid(External)

1 Container 05 467 578 001

MagNA Pure LC RNA Tissue LysisBuffer/Refill

1 Bottle 03 604 721 001

MagNA Pure DNA Tissue LysisBuffer

100 ml 04 805 160 001

Proteinase K (solution) 5 ml25 ml

03 115 828 00103 115 844 001

Disposables MagNA Pure 96 Tips (1000 l) 3840 (480 8) 05 392 900 001

MagNA Pure 96 ProcessingCartridge

36 05 435 315 001
http://www.lightcycler.com/http://www.lightcycler.com/


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6.4 Trademarks

LIGHTCYCLER, LC, MAGNA PURE, MAGNA LYSER, and REALTIME READYare trademarks of Roche.

Exiqon and ProbeLibrary are registered trademarks of Exiqon A/S, Vedbaek,Denmark.

All other product names and trademarks are the property of their respectiveowners.

6.5 Disclaimer of License

This is a product licensed under patents owned by Qiagen.

6.6 Regulatory Disclaimer

For life science research only. Not for use in diagnostic procedures.

Disposables MagNA Pure 96 Sealing Foil 100 05 435 307 001

MagNA Pure 96 Output Plate 60 05 435 285 001

MagNA Lyser Green Beads 100 tubes prefilled

with ceramic beads

03 358 941 001

Product Pack Size Cat. No.


21/21

Contact and Support If you have questions or experience problems with this or any Roche AppliedScience (RAS) product, please contact our Technical Support staff. Ourscientists commit themselves to providing rapid and effective help.We also want you to contact us if you have suggestions for enhancing RASproduct performance or using our products in new or specialized ways. Such

customer information has repeatedly proven invaluable to RAS and the world-wide research community.

To ask questions, solve problems, suggest enhancements or report new appli-cations, please visit ourOnline Technical Support Site at:

www.roche-applied-science.com/support

To call, write, fax, or email us, visit the Roche Applied Science home page,www.roche-applied-science.com, and select your home country.Country-specific contact information will be displayed.On the Roche Applied Science home page select Printed Materials to find:

in-depth Technical Manuals Lab FAQS : Protocols and references for life science research our quarterly Biochemica Newsletter Material Safety Data Sheets Pack Inserts and Product Instructions

or to request hard copies of printed materials.

Roche Diagnostics GmbHRoche Applied Science68298 MannheimGermany

0612.0

5571669001
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Documents

Instruction of Use Kit