Real-Time PCR [M.Tevfik DORAK]

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REAL-TIME PCR

M. Tevfik Dorak, MD, PhD

Glossary of Terms Used in Real-Time PCR

PowerPoint Presentation on Real-Time PCR & NEW: Real-time PCR: Troubleshooting

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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 most specifically, 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 the reaction 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 can be viewed in some systems. Real-time PCR does not detect the size of the amplicon and thus does not allow the differentiation between DNA and cDNA amplification, however, it is not influenced by non-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. In comparison 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 determines how much target concentration can vary and still be quantified. A wide dynamic range means that a wide range of ratios of target and normalizer can be assayed with equal sensitivity and specificity. It follows 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 in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. A significant increase in fluorescence above the baseline value measured during the 3-15 cycles indicates the detection of accumulated PCR product.

A fixed fluorescence threshold is set significantly above the baseline that can be altered by the operator. The parameter C_T (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 10 ^oC higher) that contain a fluorescent dye usually on the 5' base, and a quenching dye (usually TAMRA or a non-fluorescent quencher (NFQ)) typically on the 3' base (TaqMan MGB probes have a NFQ and minor groove binder at the 3’ end). When irradiated, the excited fluorescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing (this is called FRET = Förster or fluorescence resonance energy transfer) ^20,21 (Hiyoshi, 1994; Chen, 1997). Thus, the close proximity of the reporter and quencher prevents emission of any fluorescence while the probe is intact. TaqMan probes are designed to anneal to an internal region of a PCR product. When the polymerase 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 activity of quencher (no FRET) and the reporter dye starts to emit fluorescence which increases in each cycle proportional to the rate of probe cleavage. Accumulation of PCR products is detected by monitoring 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 the cleavage occurs only if the probe hybridizes to the target, the origin of the detected fluorescence is specific amplification. The process of hybridization and cleavage does not interfere with the exponential accumulation of the product. One specific requirement for fluorogenic probes is that there be no G at the 5' end. A 'G' adjacent to the reporter dye quenches reporter fluorescence even after cleavage. Well-designed TaqMan probes require very little optimization. To increase specificity of a TaqMan probe and have shorter probes, MGB or locked nucleic acid (LNA) probes can be used with equal efficiency (Kutyavin, 2000; Letertre, 2003; Johnson, 2004; Ugozzoli, 2004). See Glossary for LNA, MGB, NFQ; see also a list of SNP500 Cancer Validated TaqMan Allelic Discrimination Assays).

Molecular beacons also contain fluorescent (FAM, TAMRA, TET, ROX) and quenching dyes (typically DABCYL or BHQ) at either end but they are designed to adopt a hairpin structure while free in solution to bring the fluorescent dye and the quencher in close proximity for FRET to occur. They have two arms with complementary sequences that form a very stable hybrid or stem. The close proximity of the reporter and the quencher in this hairpin configuration suppresses reporter fluorescence. When the beacon hybridizes to the target during the annealing step, the reporter dye is separated from the quencher and the reporter fluoresces (FRET does not occur). Molecular beacons remain intact during PCR and must rebind to target every cycle for fluorescence emission. This will correlate to the amount of PCR product available. All real-time PCR chemistries allow detection of multiple DNA species (multiplexing) by designing each probe/beacon with a spectrally unique fluor/quench pair, or if SYBR 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 between acceptor and donor flurophores (LightCycler probes), reporter and quencher flurophores (TaqMan probes) 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 achieved using a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration in the unhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end. The 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence 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 the amplicon production (including non-specific amplification and primer-dimer complex) by the use of a non-sequence specific fluorescent intercalating agent (SYBR-green I or ethidium bromide). It does not bind 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 for extensive 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 longer amplicons create a stronger signal (if combined with other factors, this may cause CDC camera saturation, 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 C_T 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 to detect the increase in the fluorescent signal associated with an exponential growth of PCR product during 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 the amplification 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, secondary structure and primer quality. Although valid data can be obtained that fall outside of the efficiency range, the qRT-PCR should be further optimized or alternative amplicons designed (see Efficiency Determination Page by Pfaffl). For the slope to be an indicator of real amplification (rather than signal 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 drift is characterized by gradual increase or decrease in fluorescence without amplification of the product.) The important parameter for quantitation is the C_T. The higher the initial amount of genomic DNA, the sooner accumulated product is detected in the PCR process, and the lower the C_T 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 cycle threshold (C_T) by a mathematical analysis of the growth curve. This provides better run-to-run reproducibility. A C_T value of 40 or higher means no amplification and this value cannot be included in the calculations. Besides being used for quantitation, the C_T value can be used for qualitative analysis as a pass/fail measure.

Relative gene expression comparisons work best when the gene expression of the chosen endogenous/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 of an 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-phosphate dehydrogenase) 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 used if 18S RNA will be used as a normalizer. The 18S RNA is an overabundant RNA species, and if used as a normalizer, the C_T values are likely to be around 10-12. Any sample that has 18S C_T 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 mRNA population. Since it is abundantly expressed, 18S rRNA yields very small (<15) C_T values, which is not desirable, especially when the target gene has a low expression level. This problem can be overcome by using Ambion’s QuantumRNA™ Technology. Since the chosen mRNA species should be proportional to the amount of input RNA, it may be best to use a combination as normalizer. It is desirable to validate the chosen normalizer for the target cell or tissue. It should be expressed at a constant level at different time points by the same individual and also by different individuals at the target cell or tissue (for example, peripheral blood lymphocytes) ⁴⁰ (Dheda, 2004). This aim can be achieved by the ABI TaqMan Human Endogenous Control Plate, geNorm kit or TATAA Biocenter Endogenous Control Gene Panel which evaluate the expression of select housekeeping genes. Our own experience ⁴³ (Sabek, 2002) showed that b-actin or 18S RNA are reasonable choices as normalizers for the peripheral blood mononuclear cells, whereas GAPDH performed worst in transplant monitoring studies. Similar concerns on the choice of normalizers in transplant monitoring have also been expressed by others ⁴⁴ (Gibbs, 2003). Surveys of tumor cell lines or tissues reported the worst results with GAPDH while beta-glucuronidase (GUS) and 18S rRNA ⁴¹ (Aerts, 2004) or HPRT ⁴⁵ (de Kok, 2004) were the best choices for this target. It is important to choose a normalizer whose expression will remain constant under the experimental conditions designed for the target gene ⁴⁶ (Schmittgen, 2000; Dheda, 2005). Another study found that for robust conclusions the use of multiple internal controls is the best choice (18S rRNA and cyclophilin) for kidney mRNA expression studies ⁴⁷ (Schmid, 2003). The strategy of using multiple normalizer genes depending on the cell and tissue type is validated for general use ⁴⁸ (Vandesompele, 2002). (See Ambion: 18S RNA as an Internal Control; Ambion: GAPDH, b-actin, cyclophilin, 18S RNA as internal controls; EXPOLDB: The most constantly expressed housekeeping genes; algorithms to select the best endogenous controls: geNORM (Vandesompele, 2002), NormFinder (Andersen, 2004), and qBasePlus (Hellemans, 2007)). See also GeneInvestigator for normalizer selection (Hruz, 2011).

Multiplex TaqMan assays can be performed using multiple dyes with distinct emission wavelengths. Available dyes for this purpose are FAM, TET, VIC and JOE (the most expensive). TAMRA is reserved as the quencher on the traditional TaqMan probes and ROX as the passive reference. For best results, the combination of FAM (target) and VIC (endogenous control) is recommended (they have the largest difference in emission maximum) whereas JOE and VIC should not be combined. It is important that if the dye layer has not been chosen correctly, the machine will still read the other dye's spectrum. For example, both VIC and FAM emit fluorescence in a similar range to each other and when doing a single dye, the wells should be labeled correctly. In the case of multiplexing, the spectral compensation for the post run analysis should be turned on (on ABI 7700: Instrument/Diagnostics/Advanced Options/Miscellaneous). Activating spectral compensation improves dye spectral resolution.

One-step real-time RT-PCR performs reverse transcription and PCR in a single buffer system and in one tube. In two-step RT-PCR, these two steps are performed separately in different tubes.

TaqMan primer and probe design guidelines

1. The Primer Express software designs primers with a melting temperature (Tm) of 58-60⁰ C, and probes with a Tm value of 10⁰ C higher. The Tm of both primers should be equal,

2. Primers should be 15-30 bases in length (protocol),

3. The G+C content should ideally be 30-80%. If a higher G+C content is unavoidable, the use of high annealing and melting temperatures, cosolvents such as glycerol, DMSO, or 7-deaza-dGTP may be necessary,

4. The run of an identical nucleotide should be avoided. This is especially true for G, where runs of four or more Gs is not allowed,

5. The total number of Gs and Cs in the last five nucleotides at the 3' end of the primer should not exceed two (the newer version of the software has an option to do this automatically but not the original version). This helps to introduce relative instability to the 3' end of primers to reduce non-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 give more consistent results because PCR is more efficient and more tolerant of reaction conditions (the short 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. The higher number of Cs produces a higher DRn (this feature may require manual check). The choice of probe should be made first,

8. To avoid false-positive results due to amplification of contaminating genomic DNA in the cDNA preparation, 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 (the polymorphic 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 is efficiently degraded by AmpErase UNG (mentioned in p.9 of the manual for EZ RT-PCR kit; P/N 402877). If primers cannot be selected with dA nucleotides near the ends, the use of primers with 3' terminal dU-nucleotides should be considered.

General recommendations for real-time RT-PCR

1. 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 to 1 copy of the genome). The number of copies of total RNA used in the reaction should ideally be enough to give a signal between 20 and 30 cycles (preferably less than 100 ng) and not before 15 cycles. 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 for traditional PCR). It is optimized as 5.5 mM for the primers/probes designed using the Primer Express software. Mg concentration is usually not an issue for singleplex reactions but optimization may be important for multiplex reactions -which requires higher magnesium concentration,

ii. Concentrations of dNTPs should be balanced with the exception of dUTP (if used). Substitution of dUTP for dTTP for control of PCR product carryover requires twice dUTP that of other dNTPs. While the 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 amount by 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 TaqMan primers) and at each combination of three concentrations (50, 300, 900 nM). This means setting up three 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 target template. If necessary, a second round of optimization may improve the results. Optimal performance is achieved by selecting the primer concentrations that provide the lowest C_T 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 (<400bp) amplicons). Note that more recently developed enzymes can achieve denaturation and annealing/extension in as short as 2 seconds each (see for example Bio-Rad SsoFast Supermix). A typical reverse transcription cycle (for cDNA synthesis), which should precede the TaqMan reaction if the starting material is total RNA, consists of 10 min at 25⁰ C (primer incubation), 30 min at 48⁰ C (reverse transcription with conventional reverse 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 of carry-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 not be used with one-step RT-PCR unless rTth DNA polymerase is being used for reverse transcription and PCR (TaqMan EZ RT-PCR kit),

6. It is necessary to include at least three No Amplification Controls (NAC, a minus-reverse transcriptase control) as well as three No Template Controls (NTC, a minus sample control) in each reaction plate (to achieve a 99.7% confidence level in the definition of +/- thresholds for the target amplification, six replicates of NTCs must be run). NAC is a mock reverse transcription containing all the RT-PCR reagents, except the reverse transcriptase; NTC includes all of the RT-PCR reagents except the RNA template. It is necessary to rule out the presence of fluorescence contaminants in the sample or in the heat block of the thermal cycler (these would cause false positives). No product should be synthesized in the NTC or NAC; if a product is amplified, it indicates that one or more of the RT-PCR reagents is contaminated with DNA which may be the amplicon. If the absolute fluorescence of the NAC is greater than that of the NTC after PCR, fluorescent contaminants may be present in the sample 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 DDC_T method is going to be used for relative quantitation. The linear dynamic range refers to the range of initial template concentrations over which accurate C_T 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 C_T values (standard curve) should be a (near) straight line for both the target and normalizer real-time PCRs for the same range of 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 (even better if < 0.167; good precision), y-intercept around 40 (good sensitivity)] (see also Efficiency and Standard Curve in Glossary),

8. The passive reference is a dye (ROX) included in the reaction for ABI instruments (present in the TaqMan universal PCR master mix). It does not participate in the 5' nuclease reaction. It provides an internal reference for background fluorescence emission. This is used to normalize the reporter-dye signal. 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 for the amount of cDNA or efficiency of the PCR. Normalization is achieved by dividing the emission intensity of reporter dye by the emission intensity of the passive reference. This gives the ratio defined as Rn (further normalization by subtraction of baseline fluorescence from this value yields DRn). Not using ROX or not designating it as the passive reference dye in the analysis may cause trailing of the clusters in the allelic discrimination plot (if your instrument does not require the use of ROX -like Stratagene and Bio-Rad instruments- then ROX concentration in the master mix should not be greater than 30nM),

9. In addition to the use of ROX in ABI instruments, a master mix should be used when setting up multiple 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 the reaction before the other target gets a chance to amplify. To avoid this, the primer concentrations for the 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, the experimental samples should yield a sharp peak (first derivative plot) at the melting temperature of the amplicon (always > 80^oC), 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 direct measure of accumulation of the product of interest. If the dissociation curve has a series of peaks (usually < 80^oC), there is not enough discrimination between specific and non-specific reaction products. 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 template quality should be checked for sufficient quality and uniformity.

Recommendations for the general assay of cDNA samples

1. 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 kilobases upstream 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 not have 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 DNA contamination. Even the best RNA extraction methods yield some genomic DNA. Of course, it is ideal to 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 mixture itself (before loading) should be vortexed and mixed well. Otherwise there may be shifting Rn values during the early (0 - 5) cycles of PCR. It is also important to add probe to the buffer component and allow 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 pmol which 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 diluted 1/100, 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 for recommended 500 nM final concentration). The Applied Biosystems pre-developed TaqMan assay reagents (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 not require inclusion of a passive reference dye in the master mix, make sure your master mix contains no 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 to 250 nM for probes) are for single targets. Optimization is required for multiplex reactions, which may be 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 for example Qiagen multiplex real-time PCR kits).

Setting up one-step TaqMan reaction

One-step real-time PCR uses RNA (as opposed to cDNA) as a template. This is the preferred method if the RNA solution has a low concentration. The disadvantage is that RNA carryover prevention enzyme AmpErase cannot be used in one-step reaction format. In this method, both reverse transcription and real-time PCR take place in the same tube. The downstream PCR primer also acts as the primer for reverse transcriptase (random hexamers or oligo-dT cannot be used for reverse transcription in one-step RT-PCR). One-step reaction requires higher dNTP concentration (³ 300 mM vs 200 mM) as it combines two reactions needing dNTPs in one. A typical reaction mix for one-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 (equivalent to one diploid genome). Note that decreasing the amount of template from 100 ng to 50 ng will increase the C_T value by 1. To decrease a C_T 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 maximum amount of RNA that can be used is 1 microgram. Beware of stochastic nature of the PCR mechanics for very low template amounts. For routine analysis, 10 pg - 100 ng RNA and 100 pg - 1 mg genomic DNA can be used. See also protocols for one-step RT-PCR by Qiagen and two-step RT-PCR by Qiagen.

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

Make sure the following before starting a run:

1. Cycle parameters are correct for the run (somebody may have used different parameters before you),

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). This may have to be manually assigned after a run if the data is absent in the amplification plot but visible in 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 is labeled 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 of the 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 Run button. You will have problems and if you need to switch off and on the machine, you have to wait for at least an hour to restart the run.

When analyzing the data, remember that the default setting for baseline fluorescence calculation is cycles 3 - 15 (called baseline cycles). If any C_T value is <15 (as happens when 18S RNA is used as the normalizer), the baseline cycle range should be changed accordingly (to obtain accurate C_T values, the baseline end cycle needs to be set to start after the initial peak fluorescence and end two cycles before the C_T value for the most abundant sample). Threshold default is 10 standard deviations of the background signal above mean fluorescence generated during baseline cycles in ABI instruments. This threshold value will be used to calculate the C_T values for each sample in the run. For a useful discussion of this matter, see the ABI Tutorial on Setting Baselines and Thresholds and Real-Time PCR: Understanding C_T. (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 during the run. Saturation of CDC camera may be prevented by using optical caps rather than optical adhesive cover. It is also more likely to happen when SYBR Green I is used, when multiplexing and when a high concentration of probe is used.

For manuals and other educational material about Idaho Technology instruments, see Support page. See also a qPCR Protocol for SmartCycler at Qiagen website, and another Protocol for SmartCycler II; Critical Factors for Successful Real-time PCR by Qiagen.

Interpretation of results

At the end of each reaction, the recorded fluorescence intensity is used for the following calculations by the software of the system used:

Rn⁺ is the Rn value of a reaction containing all components (the sample of interest); Rn^- is the Rn value 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 amplification curves and gives the C_T 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 of the transcript of interest, usually by relating the PCR signal to a standard curve. In this method, a standard curve is first constructed from an RNA of known concentration. This curve is then used as a reference standard for extrapolating quantitative information for mRNA targets of unknown concentrations. 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 Quantification Page by Pfaffl.

2. Relative standard method (relative fold change): In this method, one of the experimental samples is the calibrator, or 1x sample. Each of the normalized target values is divided by the calibrator normalized target value to generate the relative expression levels. Target quantity is determined from the standard curve and divided by the target quantity of the calibrator. The calibrator is 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 are those collected after treatment or some intervention. The calibrator should be available at large enough quantities to be included in each run. See Relative Quantification Page by Pfaffl.

3. Comparative threshold (C_T) method (DDC_T): This method uses no known amount of standard but compares the relative amount of the target sequence to any of the reference values chosen and the result is given as relative to the reference value (such as the expression level of resting lymphocytes or a standard cell line or in comparison to the baseline value). For the C_T calculation to be valid, the efficiency of the target amplification and the efficiency of the reference amplification must be approximately equal. A sensitive method for assessing if two amplicons have the same efficiency is to look at how C_T varies with template dilution. Before using the DDC_T method for quantitation, a validation experiment is performed to demonstrate that efficiencies of target and reference are approximately equal. Serial dilutions of the target and normalizer are prepared and real-time PCR is run in separate tubes. The C_T values for each dilution of the target and the normalizer are obtained and their difference for each dilution is calculated (DC_T). Then, a plot of log input (like from 0.01 ng to 100 ng) amount versus DC_T is prepared (minimum three different dilutions). If the efficiencies of the two amplicons are approximately equal, the plot of log input amount versus DC_T has a slope of approximately zero (the absolute value of the slope of log input amount vs C_T 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 details of quantitation methods.

The comparative C_T method (DDC_T) for relative quantitation of gene expression

This method enables relative quantitation of template and increases sample throughput by eliminating the 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 reference should be similar. A sensitive method to control this is to look at how DC_T (the difference between the two CT values of two PCRs for the same initial template amount) varies with template dilution. If the efficiencies of the two amplicons are approximately equal, the plot of log input amount versus DC_T will have a nearly horizontal line (a slope of <0.10). This means that both PCRs perform equally efficiently across the range of initial template amounts. If the plot shows unequal efficiency, the standard curve method should be used for quantitation of gene expression. The dynamic range should be determined for both (1) minimum and maximum concentrations of the targets for which the results are accurate and (2) minimum and maximum ratios of two gene quantities for which the results are accurate. In conventional competitive RT-PCR, the dynamic range is limited to a target-to-competitor ratio of about 10:1 to 1:10 (the best accuracy is obtained for 1:1 ratio). The real-time PCR is able to achieve a much wider dynamic range.

Running the target and endogenous control amplifications in separate tubes and using the standard curve method requires the least amount of optimization and validation. The advantage of using the comparative C_T method is that the need for a standard curve is eliminated (more wells are available for samples). It also eliminates the adverse effect of any dilution errors made in creating the standard curve samples.

As long as the target and normalizer have similar dynamic ranges, the comparative C_T method (DDC_T method) is the most practical method. It is expected that the normalizer will have a higher expression level than the target (thus, a smaller C_T value). The calculations for the quantitation start with getting the difference (DC_T) between the C_T values of the target and the normalizer:

DC_T= C_T (target) - C_T (normalizer/calibrator/reference)

This value is calculated for each sample to be quantitated (unless, the target is expressed at a higher level than the normalizer, this should be a positive value. It is no harm if it is negative). One of these samples should be chosen as the reference (baseline) for each comparison to be made. The comparative DDC_T calculation involves finding the difference between each sample's DC_T and the baseline's DC_T. If the baseline value is representing the minimum level of expression, the DDC_T values are expected to be negative (because the DC_T for the baseline sample will be the largest as it will have the greatest C_T value). If the expression is increased in some samples and decreased in others, the DDC_T values will be a mixture of negative and positive ones. The last step in quantitation is to transform these values to absolute values. The formula for this (assuming 100% efficiency or doubling of the product at each cycle) is:

comparative expression level = 2 ^-^DD^Ct

For expressions increased compared to the baseline level this will be something like 2³ = 8 times 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 C_T values (there is an online ABI tutorial on the use of spread sheet programs to produce amplification plots; the TaqMan Human Endogenous Control Plate protocol also contains detailed instructions on using MS Excel for real-time PCR data analysis). Statistical assessment of the difference of DDC_T values from 0 can be achieved by a number of methods, the simplest being the t-test and Wilcoxon test (Yuan, 2006). A more 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 most useful 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 labeled with 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 before the run,

6. Normalization for the fluorescent fluctuation by using a passive reference (ROX) in the reaction and for the amount of cDNA/PCR efficiency by using an endogenous control (such as GAPDH, active reference) are different processes.

7. Real-time PCR can be used not only for qPCR but also for end-point PCR. The latter includes presence/absence assays (as in pathogen detection) and allelic discrimination assays (SNP genotyping) (see ABI User Guide),

8. Shifting Rn values during the early cycles (cycle 0-5) of PCR means initial disequilibrium of the reaction components and does not affect the final results as long as the lower value of baseline range is reset,

9. If an abnormal amplification plot has been noted (C_T value <15 cycles with amplification signal detected in early cycles), the upper value of the baseline range should be lowered and the samples should be diluted to increase the C_T value (a high C_T value may also be due to contamination),

10. A small DRn value (or greater than expected C_T value) indicates either poor PCR efficiency or low copy number of the target. This may also occur in the case of contamination of NTC,

11. A standard deviation >0.16 for C_T value indicates inaccurate pipetting,

12. SYBR Green entry in the Pure Dye Setup should be abbreviated as "SYBR" in capitals. Any other abbreviation 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 times is 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 "Auto Download" 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 by Bonetta, 2005; Biocompare; Gene-Quantification Site).

See also qPCR tips and troubleshooting at qPCR Troubleshooting Guide by ABgene; Protocol Online 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 amplicons generated

* 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 Applications

Real-Time PCR can be applied to traditional PCR applications as well as new applications that would have been less effective with traditional PCR. With the ability to collect data in the exponential growth phase, the power of PCR has been expanded into applications such as:

* Copy number variation (CNV): (Wu, 2007; ABI TaqMan® Gene Copy Number Assays; Protocol for 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/beacons ^{11,17,124-127} (Tapp, 2000; Mhlanga, 2001; Solinas, 2001; Song, 2002; Gupta, 2004; reviewed in Lareu, 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 target numbers 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 Papers

MIQE: Minimum Information for Publication of qPCR Experiments (Checklist: XLS, PDF) - Bustin, 2009 / Williams, 2012

BioTechniques 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 links

Idaho Technology LightScanner & FilmArray

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

Cepheid: Smart Cycler / GeneXpert Eppendorf Mastercycler