Real-time Pcr Em Inglês

10/04/2015 Real-Time PCR [M.Tevfik DORAK]

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Genetics Evolution HLA MHC Biostatistics Epidemiology Glossary Homepage

REAL-TIME PCRM. Tevfik Dorak, MD, PhD

Glossary of Terms Used in Real-Time PCR PowerPoint Presentation on Real-Time PCR & NEW: Real-time PCR: Troubleshooting

Webinars on qPCR (1) & (2)

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***** MIQE: Minimum Information for Publication of qPCR Experiments (Checklist: XLS, PDF) -Bustin, 2009 *****

Real-time reverse-transcriptase (RT) PCR quantitates the initial amount of the template mostspecifically, sensitively and reproducibly, and is a preferable alternative to other forms of quantitative

RT-PCR that detect the amount of final amplified product at the end-point 1 2 (Freeman, 1999;Raeymaekers, 2000; Espy, 2006). Real-time PCR monitors the fluorescence emitted during thereaction as an indicator of amplicon production during each PCR cycle (ie, in real time) as opposed to

the endpoint detection 3,4 (Higuchi, 1992; Higuchi, 1993). The real-time progress of the reaction canbe viewed in some systems. Real-time PCR does not detect the size of the amplicon and thus doesnot allow the differentiation between DNA and cDNA amplification, however, it is not influenced bynon-specific amplification unless SYBR Green is used (see below). Real-time PCR quantitation(qPCR) eliminates post-PCR processing of PCR products (which is necessary in competitive RT-PCR). This helps to increase throughput and reduce the chances of carryover contamination. Incomparison to conventional RT-PCR, real-time PCR also offers a much wider dynamic range of up to

107-fold (compared to 1000-fold in conventional RT-PCR). Dynamic range of any assay determineshow much target concentration can vary and still be quantified. A wide dynamic range means that awide range of ratios of target and normalizer can be assayed with equal sensitivity and specificity. Itfollows that the broader the dynamic range, the more accurate the quantitation.

The real-time PCR system is based on the detection and quantitation of a fluorescent reporter 5,6

(Lee, 1993; Livak, 1995). This signal increases in direct proportion to the amount of PCR product ina reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitorthe PCR reaction during exponential phase where the first significant increase in the amount of PCRproduct correlates to the initial amount of target template. The higher the starting copy number of thenucleic acid target, the sooner a significant increase in fluorescence is observed. A significantincrease in fluorescence above the baseline value measured during the 3-15 cycles indicates thedetection of accumulated PCR product. A fixed fluorescence threshold is set significantly above the baseline that can be altered by theoperator. The parameter CT (threshold cycle) is defined as the cycle number at which the

fluorescence emission exceeds the fixed threshold. There are three main fluorescence-monitoring

systems for DNA amplification 7 (Wittwer, 1997a): (1) hydrolysis probes; (2) hybridizing probes (see

Hybridization Probe Chemistry); and (3) DNA-binding agents 8,9 (Wittwer, 1997b; van der

Velden, 2003). Hydrolysis probes include TaqMan probes 10 (Heid, 1996), molecular beacons 11-

15 (Mhlanga, 2001; Vet, 2002; Abravaya, 2003; Tan, 2004; Vet & Marras, 2005) and scorpions

(further details) 16-18 (Saha, 2001; Solinas, 2001; Terry, 2002). They use the fluorogenic 5'exonuclease activity of Taq polymerase to measure the amount of target sequences in cDNA

samples (see also 19 Svanvik, 2000 for light-up probes).



TaqMan probes are oligonucleotides longer than the primers (20-30 bases long with a Tm value of 10oC higher) that contain a fluorescent dye usually on the 5' base, and a quenching dye (usually TAMRAor a non-fluorescent quencher (NFQ)) typically on the 3' base (TaqMan MGB probes have a NFQ andminor groove binder at the 3 end). When irradiated, the excited fluorescent dye transfers energy tothe nearby quenching dye molecule rather than fluorescing (this is called FRET = Frster or

fluorescence resonance energy transfer) 20,21 (Hiyoshi, 1994; Chen, 1997). Thus, the closeproximity of the reporter and quencher prevents emission of any fluorescence while the probe isintact. TaqMan probes are designed to anneal to an internal region of a PCR product. When thepolymerase replicates a template on which a TaqMan probe is bound, its 5' exonuclease activity

cleaves the 5 end of probe which contains the reporter dye 22 (Holland, 1991). This ends the activityof quencher (no FRET) and the reporter dye starts to emit fluorescence which increases in eachcycle proportional to the rate of probe cleavage. Accumulation of PCR products is detected bymonitoring the increase in fluorescence of the reporter dye (note that primers are not labeled).TaqMan assay uses universal thermal cycling parameters and PCR reaction conditions. Because thecleavage occurs only if the probe hybridizes to the target, the origin of the detected fluorescence isspecific amplification. The process of hybridization and cleavage does not interfere with theexponential accumulation of the product. One specific requirement for fluorogenic probes is that therebe no G at the 5' end. A 'G' adjacent to the reporter dye quenches reporter fluorescence even aftercleavage. Well-designed TaqMan probes require very little optimization. To increase specificity of aTaqMan probe and have shorter probes, MGB or locked nucleic acid (LNA) probes can be used withequal efficiency (Kutyavin, 2000; Letertre, 2003; Johnson, 2004; Ugozzoli, 2004). See Glossaryfor LNA, MGB, NFQ; see also a list of SNP500 Cancer Validated TaqMan Allelic DiscriminationAssays). Molecular beacons also contain fluorescent (FAM, TAMRA, TET, ROX) and quenching dyes (typicallyDABCYL or BHQ) at either end but they are designed to adopt a hairpin structure while free in solutionto bring the fluorescent dye and the quencher in close proximity for FRET to occur. They have twoarms with complementary sequences that form a very stable hybrid or stem. The close proximity ofthe reporter and the quencher in this hairpin configuration suppresses reporter fluorescence. Whenthe beacon hybridizes to the target during the annealing step, the reporter dye is separated from thequencher and the reporter fluoresces (FRET does not occur). Molecular beacons remain intact duringPCR and must rebind to target every cycle for fluorescence emission. This will correlate to theamount of PCR product available. All real-time PCR chemistries allow detection of multiple DNAspecies (multiplexing) by designing each probe/beacon with a spectrally unique fluor/quench pair, or ifSYBR green is used by melting curve analysis. By multiplexing, the target(s) and endogenous control

can be amplified in single tube for qPCR purposes. For examples, see 23-31 (Bernard, 1998; Vet,1999; Lee, 1999; Donohoe, 2000; Read, 2001; Grace, 2003; Vrettou, 2004; Rickert, 2004;Persson, 2005. Consideration of excitation, emission and absorption spectral relationships betweenacceptor and donor flurophores (LightCycler probes), reporter and quencher flurophores (TaqManprobes) and multiplexing is important and discussed in BHQ Brochure (Biosearch Technologies)and Marras SA, 2006. With Scorpion primer/probes, sequence-specific priming and PCR product detection is achievedusing a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration in theunhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled tothe 3' end. The 3' portion of the stem also contains sequence that is complementary to the extensionproduct of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiablemonomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to itscomplement within the extended amplicon thus opening up the hairpin loop. This prevents thefluorescence from being quenched and a signal is observed (see also How It Works). The cheaper alternative is the double-stranded DNA binding dye chemistry, which quantitates theamplicon production (including non-specific amplification and primer-dimer complex) by the use of anon-sequence specific fluorescent intercalating agent (SYBR-green I or ethidium bromide). It does notbind to ssDNA. SYBR green is a fluorogenic minor groove binding dye that exhibits little fluorescence



when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA 32

(Morrison, 1998). Disadvantages of SYBR green-based real-time PCR include the requirement forextensive optimization. Furthermore, non-specific amplifications require follow-up assays (melting

point or dissociation curve analysis) for amplicon identification 33 (Ririe, 1997). The method has been

used in HFE-C282Y genotyping 26 (Donohoe, 2000). Another controllable problem is that longeramplicons create a stronger signal (if combined with other factors, this may cause CDC camerasaturation, see below). Normally SYBR green is used in singleplex reactions, however when coupled

with melting curve analysis, it can be used for multiplex reactions 34 (Siraj, 2002). The threshold cycle or the CT value is the cycle at which a significant increase in DRn is first detected

(for definition of DRn, see below and Glossary). The threshold cycle is when the system begins todetect the increase in the fluorescent signal associated with an exponential growth of PCR productduring the log-linear phase. This phase provides the most useful information about the reaction(certainly more important than the end-point). The slope of the log-linear phase reflects theamplification efficiency (Eff). Eff can be calculated by the formula:

Eff = 10(-1/slope) 1 The efficiency of the PCR should be 90 - 100% (-3.6 > slope > -3.1) (see Agilent Efficiency

Calculator from the Slope). A number of variables can affect the efficiency of the PCR 35-37

(Bustin, 2004; Wong, 2005; Yuan, 2006). These factors include length of the amplicon, secondarystructure and primer quality. Although valid data can be obtained that fall outside of the efficiencyrange, the qRT-PCR should be further optimized or alternative amplicons designed (see EfficiencyDetermination Page by Pfaffl). For the slope to be an indicator of real amplification (rather thansignal drift), there has to be an inflection point. This is the point on the growth curve when the log-linear phase begins. It also represents the greatest rate of change along the growth curve. (Signal driftis characterized by gradual increase or decrease in fluorescence without amplification of the product.)The important parameter for quantitation is the CT. The higher the initial amount of genomic DNA, the

sooner accumulated product is detected in the PCR process, and the lower the CT value. The

threshold should be placed above any baseline activity and within the exponential increase phase(which looks linear in the log transformation). Some software allows determination of the cyclethreshold (CT) by a mathematical analysis of the growth curve. This provides better run-to-run

reproducibility. A CT value of 40 or higher means no amplification and this value cannot be included in

the calculations. Besides being used for quantitation, the CT value can be used for qualitative analysis

as a pass/fail measure. Relative gene expression comparisons work best when the gene expression of the chosenendogenous/internal control is more abundant and remains constant, in proportion to total RNA,among the samples. By using an invariant endogenous control as an active reference, quantitation ofan mRNA target can be normalized for differences in the amount of total RNA added to each reaction.For this purpose, the most common choices are 18S rRNA, GAPDH (glyceraldehyde-3-phosphatedehydrogenase) and b-actin, but not necessarily the most suitable choices (Radonic, 2004).Because the 18S RNA does not have a poly-A tail, cDNA synthesis using oligo-dT should not be usedif 18S RNA will be used as a normalizer. The 18S RNA is an overabundant RNA species, and if usedas a normalizer, the CT values are likely to be around 10-12. Any sample that has 18S CT values >15

may be considered poor quality. The issue of the choice of a normalizer has been reviewed by Suzuki

et al. 38 (Suzuki, 2000). The authors recommend caution in the use of GAPDH as a normalizer as it

has been shown that its expression may be upregulated in proliferating cells. They recommend b-actin as a better active reference (but see Dheda, 2004). GAPDH is severely criticized as a

normalizer by others too 39-41 (Bustin SA, 2000; Dheda, 2004; Aerts, 2004). GAPDH is particularly

an unpopular choice in cancer studies because of its increased expression in aggressive cancers 42

(Goidin, 2001). Caution should also be exercised when 18S rRNA is used as a normalizer as it is a



ribosomal RNA species (not mRNA) and may not always represent the overall cellular mRNApopulation. Since it is abundantly expressed, 18S rRNA yields very small (



specific priming. The primer conditions are the same for SYBR Green assays,6. Maximum amplicon size should not exceed 400 bp (ideally 50-150 bases). Smaller amplicons givemore consistent results because PCR is more efficient and more tolerant of reaction conditions (theshort length requirement has nothing to do with the efficiency of 5' nuclease activity),7. The probes should not have runs of identical nucleotides (especially four or more consecutive Gs),G+C content should be 30-80%, there should be more Cs than Gs, and not a G at the 5' end. Thehigher number of Cs produces a higher DRn (this feature may require manual check). The choice ofprobe should be made first,8. To avoid false-positive results due to amplification of contaminating genomic DNA in the cDNApreparation, it is preferable to have primers spanning exon-exon junctions in the cDNA sequence.This way, genomic DNA will not be amplified,9. If a TaqMan probe is designed for allelic discrimination, the mismatching nucleotide (thepolymorphic site) should be in the middle of the probe rather than at the ends,10. Use primers that contain dA nucleotides near the 3' ends so that any primer-dimer generated isefficiently degraded by AmpErase UNG (mentioned in p.9 of the manual for EZ RT-PCR kit; P/N402877). If primers cannot be selected with dA nucleotides near the ends, the use of primers with 3'terminal dU-nucleotides should be considered.See also ABgene Dual Labeled Probe Design Guide.

General recommendations for real-time RT-PCR1. Use positive-displacement pipettes to avoid inaccuracies in pipeting,2. The sensitivity of real-time PCR allows detection of the target in 3.08 pg of total RNA (equivalent to1 copy of the genome). The number of copies of total RNA used in the reaction should ideally beenough to give a signal between 20 and 30 cycles (preferably less than 100 ng) and not before 15cycles. The amount used should be decreased or increased to achieve this,3. The optimal concentrations of the reagents are as follows:i. Magnesium chloride concentration should be between 4 and 7 mM (much hogher then needed fortraditional PCR). It is optimized as 5.5 mM for the primers/probes designed using the Primer Expresssoftware. Mg concentration is usually not an issue for singleplex reactions but optimization may beimportant for multiplex reactions -which requires higher magnesium concentration,ii. Concentrations of dNTPs should be balanced with the exception of dUTP (if used). Substitution ofdUTP for dTTP for control of PCR product carryover requires twice dUTP that of other dNTPs. Whilethe optimal range for dNTPs is 500 mM to 1 mM (for one-step RT-PCR), for a typical TaqMan reaction

(PCR only), 200 mM of each dNTP (400 mM of dUTP) is used,

iii. Typically 0.25 mL (1.25 U) AmpliTaq DNA Polymerase (5.0 U/mL) is added into each 50 mL reaction.This is the minimum requirement. If necessary, optimization can be done by increasing this amountby 0.25 U increments,iv. The optimal probe concentration is 50-200 nM, and the primer concentration is 100-900 nM. Ideally,

each primer pair should be optimized at three different temperatures (58, 60 and 620 C for TaqManprimers) and at each combination of three concentrations (50, 300, 900 nM). This means setting upthree different sets (for three temperatures) with nine reactions in each (50/50 mM, 50/300 mM,50/900, 300/50, 300/300, 300/900, 900/50, 900/300, 900/900 mM) using a fixed amount of targettemplate. If necessary, a second round of optimization may improve the results. Optimal performanceis achieved by selecting the primer concentrations that provide the lowest CT and highest DRn.

Similarly, the probe concentration should be optimized for 25-225 nM,4. If AmpliTaq Gold DNA Polymerase is being used, there has to be a 9-12 min pre-PCR heat step at

92 - 950 C to activate it. If AmpliTaq Gold DNA Polymerase is used, there is no need to set up the

reaction on ice. A typical TaqMan reaction consists of 2 min at 500 C for UNG (see below) incubation,

10 min at 950 C for Polymerase activation, and 40 cycles of 15 sec at 950 C (denaturation) and 1 min

at 600 C (combined annealing and extension for small (



transcriptase) and 5 min at 950 C (reverse transcriptase inactivation),5. AmpErase uracil-N-glycosylase (UNG) is added in the reaction to prevent the reamplification ofcarry-over PCR products by removing any uracil incorporated into amplicons. This is why dUTP is

used rather than dTTP in PCR reaction. UNG does not function above 55 0C and does not cut single-

stranded DNA with terminal dU nucleotides 49 (Longo, 1990). UNG-containing master mix should notbe used with one-step RT-PCR unless rTth DNA polymerase is being used for reverse transcriptionand PCR (TaqMan EZ RT-PCR kit),6. It is necessary to include at least three No Amplification Controls (NAC, a minus-reversetranscriptase control) as well as three No Template Controls (NTC, a minus sample control) in eachreaction plate (to achieve a 99.7% confidence level in the definition of +/- thresholds for the targetamplification, six replicates of NTCs must be run). NAC is a mock reverse transcription containing allthe RT-PCR reagents, except the reverse transcriptase; NTC includes all of the RT-PCR reagentsexcept the RNA template. It is necessary to rule out the presence of fluorescence contaminants in thesample or in the heat block of the thermal cycler (these would cause false positives). No productshould be synthesized in the NTC or NAC; if a product is amplified, it indicates that one or more of theRT-PCR reagents is contaminated with DNA which may be the amplicon. If the absolute fluorescenceof the NAC is greater than that of the NTC after PCR, fluorescent contaminants may be present in thesample or in the heating block of the thermal cycler,7. The dynamic range of a primer/probe system and its normalizer should be examined if the DDCTmethod is going to be used for relative quantitation. The linear dynamic range refers to the range ofinitial template concentrations over which accurate CT values are obtained. This is determined by

running (in triplicate) reactions of five RNA concentrations (for example, 3 pg/mL, 30 pg/mL, 300 pg/mL,

3 ng/mL and 30 ng/mL). The resulting plot of log of the initial amount vs CT values (standard curve)

should be a (near) straight line for both the target and normalizer real-time PCRs for the same rangeof total RNA concentrations [ideal values are: slope = -3.32 (corresponding to 100% efficiency; 90-

110% is acceptable), R2 > 0.99 (also indicates good efficiency), standard deviation < 0.250 (evenbetter if < 0.167; good precision), y-intercept around 40 (good sensitivity)] (see also Efficiency andStandard Curve in Glossary),8. The passive reference is a dye (ROX) included in the reaction for ABI instruments (present in theTaqMan universal PCR master mix). It does not participate in the 5' nuclease reaction. It provides aninternal reference for background fluorescence emission. This is used to normalize the reporter-dyesignal. This normalization is for non-PCR-related fluorescence fluctuations occurring in different wells(concentration or volume differences, bubbles) or over time and different from the normalization forthe amount of cDNA or efficiency of the PCR. Normalization is achieved by dividing the emissionintensity of reporter dye by the emission intensity of the passive reference. This gives the ratio definedas Rn (further normalization by subtraction of baseline fluorescence from this value yields DRn). Notusing ROX or not designating it as the passive reference dye in the analysis may cause trailing of theclusters in the allelic discrimination plot (if your instrument does not require the use of ROX -likeStratagene and Bio-Rad instruments- then ROX concentration in the master mix should not begreater than 30nM),9. In addition to the use of ROX in ABI instruments, a master mix should be used when setting upmultiple reactions to minimize sample-to-sample and well-to-well variation and improve reproducibility(ROX will be within the master mix),10. If multiplexing is done, the more abundant of the targets will use up all the ingredients of thereaction before the other target gets a chance to amplify. To avoid this, the primer concentrations forthe more abundant target should be limited (and Mg concentration optimization may be necessary),11. If SYBR green is used, dissociation (melting) curve analysis should be performed. Ideally, theexperimental samples should yield a sharp peak (first derivative plot) at the melting temperature of the

amplicon (always > 80oC), whereas the NAC and NTC will not generate significant fluorescent signal.This result indicates that the products are specific, and that SYBR Green I fluorescence is a directmeasure of accumulation of the product of interest. If the dissociation curve has a series of peaks

(usually < 80oC), there is not enough discrimination between specific and non-specific reactionproducts. To obtain meaningful data, optimization of the RT-PCR would be necessary,12. Each experiment should contain proper controls (no template control, no-RT control) and templatequality should be checked for sufficient quality and uniformity.



Recommendations for the general assay of cDNA samples1. Reverse transcription of total RNA to cDNA should be done with random hexamers (not with oligo-dT). If oligo-dT has to be used long mRNA transcripts or amplicons greater than two kilobasesupstream should be avoided, and 18S RNA cannot be used as normalizer (it has no poly-A tail),2. Multiplex PCR will only work properly if the control primers are limiting (ABI control reagents do nothave their primers limited). This requires running primer limiting assays for optimization,3. The range of target cDNA used is 1 ng to 1 mg. If DNA is used (mainly for allelic discrimination

studies), the optimum amount is 100 ng to 1 mg,4. It is ideal to treat each RNA preparation with RNAse-free DNAse to avoid genomic DNAcontamination. Even the best RNA extraction methods yield some genomic DNA. Of course, it is idealto have primers not amplifying genomic DNA at all but sometimes this may not be possible,5. For optimal results, the reagents (before the preparation of the PCR mix) and the PCR mixtureitself (before loading) should be vortexed and mixed well. Otherwise there may be shifting Rn valuesduring the early (0 - 5) cycles of PCR. It is also important to add probe to the buffer component andallow it to equilibrate at room temperature prior to reagent mix formulation. TaqMan primers and probes

The TaqMan probes ordered from ABI at midi-scale arrive already resuspended at 100 mM. If a 1/20

dilution is made, this gives a 5 mM solution. This stock solution should be aliquoted, frozen and kept in

the dark. Using 1 mL of this in a 50 mL reaction gives the recommended 100 nM final concentration(the range for final probe concentration in a TaqMan reaction is 50 to 250 nM).The primers arrive lyophilized with the amount given on the tube in pmols (such as 150.000 pmolwhich is equal to 150 nmol). If X nmol of primer is resuspended in X mL of H2O, the resulting solution

is 1 mM. It is best to freeze this stock solution in aliquots. When the 1 mM stock solution is diluted1/100, the resulting working solution will be 10 mM. To get the recommended 50 - 900 nM final primer

concentration in 50 mL reaction volume, 0.25 - 4.50 mL should be used per reaction (2.5 mL forrecommended 500 nM final concentration). The Applied Biosystems pre-developed TaqMan assayreagents (PDAR) are supplied as a primer and probe mix in one tube usually at 20X or 40X colution.They have to be used as 20X, meaning 2.5 mL in a 50 mL reaction volume. If your instrument does notrequire inclusion of a passive reference dye in the master mix, make sure your master mix containsno or small amount ROX (around 30nM final concentration).The ranges for final primer and probe concentrations given here (50 to 900 nM for primers and 50 to250 nM for probes) are for single targets. Optimization is required for multiplex reactions, which maybe extensive (see QIAGEN: Critical factors for success in real-time, multiplex PCR). Alternatively,specially designed kits for multiplexing may be used to avoid extensive optimization steps (see forexample Qiagen multiplex real-time PCR kits). Setting up one-step TaqMan reactionOne-step real-time PCR uses RNA (as opposed to cDNA) as a template. This is the preferredmethod if the RNA solution has a low concentration. The disadvantage is that RNA carryoverprevention enzyme AmpErase cannot be used in one-step reaction format. In this method, bothreverse transcription and real-time PCR take place in the same tube. The downstream PCR primeralso acts as the primer for reverse transcriptase (random hexamers or oligo-dT cannot be used forreverse transcription in one-step RT-PCR). One-step reaction requires higher dNTP concentration (300 mM vs 200 mM) as it combines two reactions needing dNTPs in one. A typical reaction mix forone-step PCR by Gold RT-PCR kit is as follows:H2O + RNA : 20.5 mL (24 mL if PDAR is used)

10X TaqMan buffer : 5.0 mL

MgCl2 (25 mM) : 11.0 mL

dATP (10mM) : 1.5 mL (for final concentration of 300 mM)

dCTP (10mM) : 1.5 mL (for final concentration of 300 mM)

dGTP (10mM) : 1.5 mL (for final concentration of 300 mM)

dUTP (20mM) : 1.5 mL (for final concentration of 600 mM)



Primer F (10 mM) * : 2.5 mL (for final concentration of 500 nM)

Primer R (10 mM) * : 2.5 mL (for final concentration of 500 nM)

TaqMan Probe * : 1.0 mL (for final concentration of 100 nM)

AmpliTaq Gold : 0.25 mL (can be increased for higher efficiency)

Reverse Transcriptase : 0.25 mL

RNAse inhibitor : 1.00 mL

* If a PDAR is used, 2.5 mL of primer + probe mix used.

Ideally 100 pg - 100 ng RNA should be used in this reaction and no less than 6.6 picogram (equivalentto one diploid genome). Note that decreasing the amount of template from 100 ng to 50 ng willincrease the CT value by 1. To decrease a CT value by 3, the initial amount of template should be

increased 8-fold. ABI claims that 2 picogram RNA can be detected by this system and the maximumamount of RNA that can be used is 1 microgram. Beware of stochastic nature of the PCR mechanicsfor very low template amounts. For routine analysis, 10 pg - 100 ng RNA and 100 pg - 1 mg genomicDNA can be used. See also protocols for one-step RT-PCR by Qiagen and two-step RT-PCR byQiagen.

Cycling parameters for one-step PCR

Reverse transcription (by MuLV) 480 C for 30 min

AmpliTaq activation 950 C for 10 min

PCR: denaturation 950 C for 15 sec and annealing/extension 600 C for 1 min (repeated 40 times)(note that there are only two steps in a typical real-time PCR cycle unless amplicon size is large

(>400bp) or Tm values for the primers are high (>600 C))(On ABI 7700, minimum holding time is 15 seconds.) The EZ one-step RT-PCR kit allows the use of UNG as the incubation time for reverse transcription

is 60 0C thanks to the use of a thermostable reverse transcriptase. This temperature is also a better

option to avoid primer dimers and non-specific bindings at 48 0C (see also Roche LightCycler One-Step RT-PCR Kit).

Operating ABI 7700(See also ABI 7000 Compendium; Protocol for ABI 7500)Make sure the following before starting a run:1. Cycle parameters are correct for the run (somebody may have used different parameters beforeyou),2. Choice of spectral compensation is correct (off for singleplex, on for multiplex reactions),3. Choice of "Number of PCR Stages" is correct in the Analysis Options box (Analysis/Options). Thismay have to be manually assigned after a run if the data is absent in the amplification plot but visiblein the plate view, and the X-axis of the amplification is displaying a range of 0-1 cycles,4. No Template Control (NTC) is labeled as such (for accurate DRn calculations),5. The choice of dye component should be made correctly before data analysis. Even if the probe islabeled with FAM and VIC is chosen there will be some result but the wrong one,6. You must save the run before it starts by giving it a name (not leaving as untitled). Also at the end ofthe run, first save the data before starting to analyze,7. The ABI software requires extreme caution. Do not attempt to stop a run after clicking on the Runbutton. You will have problems and if you need to switch off and on the machine, you have to wait forat least an hour to restart the run. When analyzing the data, remember that the default setting for baseline fluorescence calculation iscycles 3 - 15 (called baseline cycles). If any CT value is



cycles before the CT value for the most abundant sample). Threshold default is 10 standard

deviations of the background signal above mean fluorescence generated during baseline cycles in ABIinstruments. This threshold value will be used to calculate the CT values for each sample in the run.

For a useful discussion of this matter, see the ABI Tutorial on Setting Baselines and Thresholdsand Real-Time PCR: Understanding CT. (Interestingly, this issue is best discussed in the manual

for TaqMan Human Endogenous Control Plate.)

If the results do not make sense, check the raw spectra for a possible CDC camera saturation duringthe run. Saturation of CDC camera may be prevented by using optical caps rather than opticaladhesive cover. It is also more likely to happen when SYBR Green I is used, when multiplexing andwhen a high concentration of probe is used.

For manuals and other educational material about Idaho Technology instruments, see Supportpage. See also a qPCR Protocol for SmartCycler at Qiagen website, and another Protocol forSmartCycler II; Critical Factors for Successful Real-time PCR by Qiagen. Interpretation of resultsAt the end of each reaction, the recorded fluorescence intensity is used for the following calculationsby the software of the system used:

Rn+ is the Rn value of a reaction containing all components (the sample of interest); Rn- is the Rnvalue detected in NTC (baseline value). DRn is the difference between Rn+ and Rn-. It is an indicator

of the magnitude of the signal generated by the PCR (DRn may be called RFU=relative fluorescence

unit in some instruments). It is the DRn plotted against cycle numbers that produces the amplificationcurves and gives the CT value.

There are different approaches to quantitate the amount of template 50 (Livak, 2001):1. Absolute standard curve method: Absolute quantification determines the input copy number ofthe transcript of interest, usually by relating the PCR signal to a standard curve. In this method, astandard curve is first constructed from an RNA of known concentration. This curve is then used as areference standard for extrapolating quantitative information for mRNA targets of unknownconcentrations. cDNA plasmids are the preferred standards for absolute quantitation. This method

has been used to estimate cytokine concentrations 51 (Giulietti, 2001), CMV 52-55 (Kearns, 2001a;

Kearns, 2001b; Kearns, 2002; Mengelle, 2003), HIV 56 (Gibellini, 2004) and other viral loads 57

(Niesters, 2001), 16 (Saha, 2001). See Bustin, 2000 for a review 39; and Absolute QuantificationPage by Pfaffl.2. Relative standard method (relative fold change): In this method, one of the experimentalsamples is the calibrator, or 1x sample. Each of the normalized target values is divided by thecalibrator normalized target value to generate the relative expression levels. Target quantity isdetermined from the standard curve and divided by the target quantity of the calibrator. The calibratoris the 1x sample, and all other quantities are expressed as an n -fold difference relative to the

calibrator. The calibrator is usually the expression level at baseline and the experimental samples arethose collected after treatment or some intervention. The calibrator should be available at largeenough quantities to be included in each run. See Relative Quantification Page by Pfaffl.

3. Comparative threshold (CT) method (DDCT): This method uses no known amount of standard

but compares the relative amount of the target sequence to any of the reference values chosen andthe result is given as relative to the reference value (such as the expression level of restinglymphocytes or a standard cell line or in comparison to the baseline value). For the CT calculation to

be valid, the efficiency of the target amplification and the efficiency of the reference amplification mustbe approximately equal. A sensitive method for assessing if two amplicons have the same efficiencyis to look at how CT varies with template dilution. Before using the DDCT method for quantitation, a

validation experiment is performed to demonstrate that efficiencies of target and reference areapproximately equal. Serial dilutions of the target and normalizer are prepared and real-time PCR isrun in separate tubes. The CT values for each dilution of the target and the normalizer are obtained



and their difference for each dilution is calculated (DCT). Then, a plot of log input (like from 0.01 ng to

100 ng) amount versus DCT is prepared (minimum three different dilutions). If the efficiencies of the

two amplicons are approximately equal, the plot of log input amount versus DCT has a slope of

approximately zero (the absolute value of the slope of log input amount vs CT should be < 0.1). This

method has been used in monitoring the immune system activity after transplantation 43 (Sabek,

2002). See Livak & Schmittgen, 2001 for a review 50; and ABI-7700 User Bulletin #2 for the detailsof quantitation methods.

The comparative CT method (DDCT) for relative quantitation of gene expression

This method enables relative quantitation of template and increases sample throughput by eliminatingthe need for standard curves when looking at expression levels relative to an active reference control(normalizer). For this method to be successful, the dynamic range of both the target and referenceshould be similar. A sensitive method to control this is to look at how DCT (the difference between the

two CT values of two PCRs for the same initial template amount) varies with template dilution. If theefficiencies of the two amplicons are approximately equal, the plot of log input amount versus DCT will

have a nearly horizontal line (a slope of



increase, and for decreased expression it will be something like 2-3 = 1/8 of the reference level.Microsoft Excel can be used to do these calculations by simply entering the CT values (there is an

online ABI tutorial on the use of spread sheet programs to produce amplification plots; the TaqManHuman Endogenous Control Plate protocol also contains detailed instructions on using MS Excel

for real-time PCR data analysis). Statistical assessment of the difference of DDCT values from 0 can

be achieved by a number of methods, the simplest being the t-test and Wilcoxon test (Yuan, 2006). Amore accurate method of relative quantification using the relative expression ratio is presented by

Pfaffl 58 (Pfaffl, 2001). The quantification methods are outlined in the ABI User Bulletins. The Bulletins #2 and #5 are mostuseful for the general understanding of real-time PCR and quantification.

Points to remember and trouble shooting

1. TaqMan Universal PCR master mix should be stored at 2 to 8 0C (not at -20 0C),2. The GAPDH probe supplied with the TaqMan Gold RT-PCR kit is labeled with a JOE reporter dye,the same probe provided within the Pre-Developed TaqMan Assay Reagents (PDAR) kit is labeledwith VIC. Primers for these human GAPDH assays are designed not to amplify genomic DNA,3. The carryover prevention enzyme, AmpErase UNG, cannot be used with one-step RT-PCR which

requires incubation at 48 0C but may be used with the EZ RT-PCR kit,4. It is ideal to run duplicates to control pipeting errors but this inevitably increases the cost,5. If multiplexing, the spectral compensation option (in Advanced Options) should be checked beforethe run,6. Normalization for the fluorescent fluctuation by using a passive reference (ROX) in the reaction andfor the amount of cDNA/PCR efficiency by using an endogenous control (such as GAPDH, activereference) are different processes.7. Real-time PCR can be used not only for qPCR but also for end-point PCR. The latter includespresence/absence assays (as in pathogen detection) and allelic discrimination assays (SNPgenotyping) (see ABI User Guide),8. Shifting Rn values during the early cycles (cycle 0-5) of PCR means initial disequilibrium of thereaction components and does not affect the final results as long as the lower value of baseline rangeis reset,9. If an abnormal amplification plot has been noted (CT value 0.16 for CT value indicates inaccurate pipetting,

12. SYBR Green entry in the Pure Dye Setup should be abbreviated as "SYBR" in capitals. Any otherabbreviation or lower case letters will cause problems,13. The ABI 7700 should not be deactivated for extended periods of time. If it has ever been shutdown,it should be allowed to warm up for at least one hour before a run. Leaving the instrument on all timesis recommended and is beneficial for the laser. If the machine has been switched on just before a run,an error box stating a firmware version conflict may appear. If this happens, choose the "AutoDownload" option,14. The ABI 7700 (or its successor 7900) is only one of the many real-time PCR systems in a very

competitive market (see reviews by Bustin SA, 2000, Bustin SA, 2002; 39,59 Supplier Guide byBonetta, 2005; Biocompare; Gene-Quantification Site). See also qPCR tips and troubleshooting at qPCR Troubleshooting Guide by ABgene; ProtocolOnline qPCR troubleshooting; Ten Most Common Real-Time PCR Pitfalls. Advantages of using Real-Time PCR* Traditional PCR is measured at end-point (plateau), while real-time PCR collects data in the



exponential growth phase* An increase in reporter fluorescent signal is directly proportional to the number of ampliconsgenerated* The cleaved probe provides a permanent record amplification of an amplicon* Increased dynamic range of detection* Requirement of 1000-fold less RNA than conventional assays* No-post PCR processing due to closed system (no electrophoretical separation of amplified DNA)* Detection is capable down to a 2-fold change* Small amplicon size results in increased amplification efficiency (even with degraded DNA) Real-Time PCR ApplicationsReal-Time PCR can be applied to traditional PCR applications as well as new applications that wouldhave been less effective with traditional PCR. With the ability to collect data in the exponential growthphase, the power of PCR has been expanded into applications such as: * Copy number variation (CNV): (Wu, 2007; ABI TaqMan Gene Copy Number Assays; Protocolfor 7900HT)

* Quantitation of gene expression (Giulietti, 2001) including NK cell KIR gene expression 60 (Leung,2005)

* Array verification 61 (Rajeevan, 2001). See also Verification of Array Results Page by Pfaffl.

* Biosafety and genetic stability testing 62 (Lovatt, 2002)

* Drug therapy efficacy / drug monitoring 63 64 65 66 (Leruez-Ville, 2004; Brennan, 2003; Burger,2003; Kogure, 2004)

* Real-Time Immuno-PCR (IPCR) 67-69 (Adler, 2003; Barletta, 2004; Lind & Kubista, 2005)

* Chromatin Immunoprecipitation (ChIP) 70-75 (Braveman, 2004; Sandoval, 2004; Wang, 2004;Iype, 2005; Potratz, 2005; Puppo, 2005)

* Viral quantitation 55,57 (Niesters, 2001; Mengelle, 2003; Espy, 2006)

* Pathogen detection 76-81 (Belgrader, 1999; Uhl, 2002; Mackay, 2004; Perandin, 2004;

Watzinger, 2004; Reynisson, 2006; Espy, 2006) including CMV detection 52-55 (Kearns, 2001a;

Kearns, 2001b; Kearns, 2002; Mengelle, 2003), rapid diagnosis of meningococcal infection 82

(Bryant, 2004), penicillin susceptibility of Streptococcus pneumoniae 83 (Kearns, 2002),

Mycobacterium tuberculosis and its resistant strains 84-87 (Kraus, 2001; Torres, 2003; Cleary, 2003;

Hazbon, 2004), and waterborne microbial pathogens in the environment 88,89 (Foulds, 2002; Guy,2003)

* Radiation exposure assessment 28,91-93 (Blakely, 2001; Blakely, 2002; Grace, 2002; Grace,2003)

* In vivo imaging of cellular processes 94,95 (Tung, 2000; Bremer, 2002)

* DNA damage (microsatellite instability) estimation 90 (Dietmaier, 2001)* DNA damage (nuclear DNA) and DNA adduct estimation: Laws, 2001; Grimaldi, 2002; Santos,2006; Meyer JN, 2010

* Mitochondrial DNA studies (CNV, damage, deletion) 96-98 (He, 2002; Liu, 2003; Alonso, 2004; Lin,2003; Santos, 2006; Lin, 2008; Edwards JG, 2009; Meyer JN, 2010; Rothfuss, 2010)

* Methylation detection 99-102 (Eads, 2000; Trinh, 2001; Cottrell, 2004; Lehmann & Kreipe, 2004;Thomassin, 2004; Holemon, 2007; Dugast-Garzaqk & Grange, 2009; Campan, 2009; Tost J (Ed),2009)* Measurement of unmethylated repeat DNA sequences (Rand & Molloy, 2010)

* Detection of inactivation at X-chromosome 103,104 (Hartshorn, 2002; van Dijk, 2002)

* Determination of identity at highly polymorphic HLA loci 105 (Zhou, 2004)

* Monitoring post transplant solid organ graft outcome 43,44 (Sabek, 2002; Gibbs, 2003)



* Monitoring chimerism after hematopoietic stem cell transplantation 106-109 (Elmaagacli, 2002;Alizadeh, 2002; Thiede, 2004; Harries, 2004)

* Monitoring minimal residual disease after hematopoietic stem cell transplantation 9,106,110-112

(Elmaagacli, 2002; Cilloni, 2002; Sarris, 2002; Gabert, 2003; Van der Velden, 2003)

* Determination of gene dosage and zygosity 113-115 (Bubner, 2004; Barrois, 2004; Chen, 2006;Szilagyi, 2006; Wu, 2007; Parajes, 2007 & 2008)* Genotyping by fluorescence melting-curve analysis (FMCA) or high-resolution melting (HRM)

analysis 26,116-123 (von Ahsen 2000; Donohoe, 2000; Lyon, 2001; Waterfall & Cobb, 2002;Bennett, 2003; Wittwer, 2003; Zhou, 2005; Palais, 2005; Chou, 2005) or specific probes/beacons11,17,124-127 (Tapp, 2000; Mhlanga, 2001; Solinas, 2001; Song, 2002; Gupta, 2004; reviewed inLareu, 2004). LNA or MGB probes can be used allelic discrimination too (Kutyavin, 2000; Letertre,2003; Johnson, 2004; Ugozzoli, 2004; Gibson NJ, 2006; Shen, 2009; Schleinitz, 2011)

- Trisomies 128 (Zimmermann, 2002) and single-gene copy numbers 129-132 (Bieche, 1998;Mocellin, 2003, Barrois, 2004; Linzmeier, 2005)

- Microdeletion genotypes 133-136 (Laurendeau, 1999; Kariyazono, 2001; Covault, 2003; Coupry,2004; Rose-Zerilli, 2009 (GST deletion))

- Haplotyping 137,138 (Von Ahsen, 2004; Pont-Kingdon & Lyon, 2005)

- Quantitative microsatellite analysis 139 (Ginzinger, 2000)

- DNA pooling and quantitative allelic discrimination 140-142 (Barcellos, 2001; Abbas, 2004;Quesada, 2004; Gibson NJ, 2006)

- Prenatal diagnosis / sex determination using single cell isolated from maternal blood 143-145

(Hahn, 2000; Bischoff, 2002; Bischoff, 2003) or fetal DNA in maternal circulation 144,146 (Bischoff,2002; Hwa, 2004)

- Prenatal diagnosis of hemoglobinopathies 29,147,148 (Kanavakis, 1997; Vrettou, 2003; Vrettou,2004)

- Intraoperative cancer diagnostics 149 (Raja, 2002)* Linear-after-the-exponential (LATE)-PCR: a new method for real-time quantitative analysis of targetnumbers in small samples, which is adaptable to high throughput applications in clinical diagnostics,

biodefense, forensics, and DNA sequencing 150 (Sanchez, 2004).

Full References Cited Automated PubMed Search for Real-Time PCR New Publications

Open Access Real-time PCR PapersMIQE: Minimum Information for Publication of qPCR Experiments (Checklist: XLS, PDF) -

Bustin, 2009 / Williams, 2012BioTechniques Molecular Biology Forums: Real-Time qPCR

Quantitative PCR Gene Expression Profiling by MultiD - Tutorials TATAA Biocenter Courses in Quantitative PCR

Workshops and Courses by Stephen Bustin

Internet linksIdaho Technology LightScanner & FilmArray

ROCHE LightCycler Online Bio-Rad: CFX96 Stratagene-Agilent qPCR System Corbett/Qiagen Rotor-Gene

Cepheid: Smart Cycler / GeneXpert Eppendorf MastercyclerApplied Biosystems Sequence Detection Systems

Bio-Rad Real-Time PCR Applications GuideCritical Factors for Successful Real-time PCR (Qiagen)



ABI User Bulletins ABI-PRISM 7700 Application Notes 7900HT 7000 Compendium SNP500Cancer Validated Allelic Discrimination Assay List (including TaqMan Protocols)

Available Real-Time PCR Platforms BioCompare Horizon Press 1st International qPCR Symposium & Application Workshop (qPCR 2009)

qPCR Guide (EuroGentec)

Scorpion Technology Molecular Beacons Light-Up Probes (1) (2) D-LUX Designer LNA Probes LNA Primers (Exiqon OligoDesign) Exiqon ProbeLibrary

DesignMyProbe at Sigma-AldrichFluidigm (high-throughput qPCR, CNV, genotyping)

TaqMan Gene Expression AssaysProducts at TATAA BioCenter & GenEx

Primer-Probe and Beacon Design Program & Demo by PremierRT-PCR PrimerBank RT-PCR Primer DataBase RT-PCR Primer Sets

AlleleID Pathogen Detection Primer & Probe Design Tool by Premier Biosoft InternationalBioSearch RealTimeDesign Software (QuickStart Guide)

Quantitative PCR Primer Database - QPPD (NCI)PrimerDesign InVitroGene: Custom Primers-OligoPerfect Designer

Frequently Asked Questions (Real-Time PCR Primers)

Applied Biosystems: Real-Time PCR Animated Tutorials (for beginners)Transcript of a Webcast on Real-Time PCR Applications (Bio.Com)

Biocompare Tutorials:Tools and Technologies for Real-Time PCR & Fast PCR (text)

Sigma-Aldrich qPCR WebinarsInvitrogene Molecular Probes Handbook

Roche LightCycler Literature and Technical NotesQiaGen Handbooks on SYBR Green Detection Systems and RT-PCR

Ambion TechNotes on Real-Time PCRFull qPCR Protocol (Nolan, Hands & Bustin, Nature Protocols, 2006) (PDF)

Gene Quantification Page by Michael W Pfaffl & Directory PageReal-Time PCR Tutorial (South Carolina University)Real-Time PCR: Short Course (University of Texas)

Real-Time PCR Handbook (University of Illinois at Chicago)Troubleshooting & Optimization Guide (Thermo Scientific)Real-Time PCR Seminar (NIEHS) & Review by Nigel Walker

Real-Time PCR in Infectious Diseases (PPT) & (PDF) by Theo SlootsReal Time PCR & Quantitation Lecture by Ian MacKay

Essentials of Real-Time PCR Lecture by Man Bock Gu Q-PCR Training @ TATAA BioCenter

PCR and Real Time PCR Links Real-Time PCR Literature Links at PCRlinks.com Links at Protocol Online

Review of real-time PCR in mRNA Quantitation (Wong & Medrano, 2005)Five Questions on qPCR & How It Works (The Scientist)

Statistics and Gene Expression Analysis BioInformatics in Real-Time PCR

qPCR Data Analysis Presentation (RB Lanz, 2008)Q-GENE for data processing

geNORM (Vandesompele, 2002) NormFinder (Andersen, 2004) qBasePlus (Hellemans, 2007)

BestKeeper for determination of stable housekeeping genes (Download)

REST for Relative Expression Software Tool (REST-2008 / Corbett)CAmpER - Real-time PCR Analysis Software

Peirson, 2003 (DART-PCR) (Download)SNPman (User Guide) for TaqMan allelic discrimination assay genotype calling



for the ABI7300, LC480 and Biorad CFX platforms (Konopac, 2011)

Real Time PCR Special Issues:METHODS: Dec 2001, Vol.25, Issue 4 & April 2010, Vol.50, Issue 4

CLINICA CHIMICA ACTA: Jan 2006, Vol.363, Issue 1-2HUMAN MUTATION: June 2009, Vol.30, Issue 6 (High-Resolution Melting Technology)

Books:

Real-Time PCR (Dorak MT)A-Z of Quantitative PCR (Bustin S)

Rapid Cycle Real-Time PCR-Methods and Applications (Wittwer Hahn, Kaul)Real-Time PCR: An Essential Guide (Edwards, Logan, Saunders)

Real-Time PCR: Current Technology and Applications (Logan, Edwards, Saunders)Real-time PCR in Microbiology (MacKay IM)

Address for bookmark: http://www.dorak.info/genetics/realtime.html

Dorak MT (Ed): Real-Time PCR (Advanced Methods Series). Oxford:Taylor & Francis, 2006

(Amazon) (Table of Contents) (Google Books)

M.Tevfik Dorak, MD, PhDLast updated on 13 November 2012

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