Genetics Real-Time PCR Homepage

GLOSSARY OF REAL-TIME PCR TERMS

M.Tevfik Dorak

Absolute quantification: The absolute quantitation assay is used to quantitate unknown samples by interpolating their quantity from a standard curve (as in determination of viral copy number). (Absolute Quantification Page by Pfaffl).

Allelic discrimination assay: Assays designed to type for gene variants. Either differentially labeled (TaqMan®) probes (one for each variant) or a single probe and melting curve analysis can be used for this purpose. Alternative methods include dsDNA-binding dyes (in combination with melting curve analysis). TaqMan®-based allelic discrimination assays are analyzed by differences in threshold cycles or by endpoint fluorescence value for each allele. The results are plotted by fluorescence intensity or by C_t values for each allele at X and Y axes (see Osgood-McWeeney, 2000 and Figures 3-5 in Hu & Chen for examples). See ABI Allelic Discrimination with TaqMan® Probes and Getting Started Guides for ABI 7000 & 7900HT, LightScanner^® and Amplifluor^® SNPs Genotyping System.

Amplicon: The amplified sequence of DNA in the PCR process.

Amplification plot: The plot of cycle number versus fluorescence signal which correlates with the initial amount of target nucleic acid during the exponential phase of PCR.

Anchor & reporter probes: Two partnering LightCycler (hybridizing) probes that hybridize on the target sequence in close proximity. The anchor probe (donor) emits fluorescence to excite the reporter probe (acceptor) to initiate FRET. In allelic discrimination assays, it is important that the reporter probe spans the mutation and has a lower Tm than the anchor probe.

Baseline: The initial cycles of PCR during which there is little change in fluorescence signal (usually cycles 3 to 15).

Baseline value: During PCR, changing reaction conditions and environment can influence fluorescence. In general, the level of fluorescence in any one well corresponds to the amount of target present. Fluorescence levels may fluctuate due to changes in the reaction medium creating a background signal. The background signal is most evident during the initial cycles of PCR prior to significant accumulation of the target amplicon. During these early PCR cycles, the background signal in all wells is used to determine the ‘baseline fluorescence’ across the entire reaction plate. The goal of data analysis is to determine when target amplification is sufficiently above the background signal, facilitating more accurate measurement of fluorescence.

Calibrator: A single reference sample used as the basis for relative-fold increase in expression studies (assuming constant reaction efficiency). This calibrator should be included in each assay.

Coefficient of variation (CV): Used as a measure of experimental variation. It is important that a linear value (e.g., copy numbers) is used to calculate the CV (but not C_t values which are logarithmic). Intra-assay CV quantifies the amount of error seen within the same assay (in duplicates) and inter-assay CV quantifies the error between separate assays.

C_t (threshold cycle): Threshold cycle reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number. The C_t value assigned to a particular well thus reflects the point during the reaction at which a sufficient number of amplicons have accumulated. Also called crossing point (C_p) in LightCycler terminology.

Derivative curve: This curve is used in Tm analysis. It has the temperature in the x axis and the negative derivative of fluorescence (F) with respect to temperature (T), shown as dF/dT, on the y axis. The reproducibility of a derivative melting curve is high with a standard deviation of only 0.1 ^oC between runs.

dsDNA-binding agent: A molecule that emits fluorescence when bound to dsDNA. The prototype is SYBR® Green I. In real-time PCR, the fluorescence intensity increases proportionally to dsDNA (amplicon) concentration. The problem with DNA-binding agents is that they bind to all dsDNA products: specific amplicon or non-specific products (misprimed targets and primer-dimers included). For this reason, analysis using DNA-binding agents is usually coupled with melting analysis.

Dynamic range: The range of initial template concentrations over which accurate C_t values are obtained. If endogenous control is used for DDC_t quantitation method, dynamic ranges of target and control should be comparable. In absolute quantitation, interpolation within this range is accurate but extrapolation beyond the dynamic range should be avoided. The larger the dynamic range, the greater the ability to detect samples with high and low copy number in the same run.

Efficiency of the reaction: The efficiency of the reaction can be calculated by the following equation: E = 10^(-1/slope) –1. The efficiency of the PCR should be 90-100% meaning doubling of the amplicon at each cycle. This corresponds to a slope of –3.1 to –3.6 in the C_t vs log-template amount standard curve. In order to obtain accurate and reproducible results, reactions should have efficiency as close to 100% as possible (e.g., two-fold increase of amplicon at each cycle), and in any case, efficiency should be similar for both target and reference (normalizer, calibrator, endogenous control, internal control). A number of variables can affect the efficiency of the PCR. These factors can include length of the amplicon, presence of inhibitors, secondary structure and primer design. Although valid data can be obtained that fall outside of the efficiency range, if it is < 0.90, the quantitative real-time PCR should be further optimized or alternative amplicons designed (see Efficiency Determination).

End-point analysis: As opposed to quantitative analysis using the data collected during exponential phase of PCR, real-time applications can also be used to collect end-point data for qualitative assays. These are either allelic discrimination assays (genotyping) or absence/presence assays (pathogen detection).

Endogenous control: This is an RNA or DNA that is naturally present in each experimental sample. By using an invariant endogenous control as an active 'reference', quantitation of a messenger RNA (mRNA) target can be normalized for differences in the amount of total RNA added to each reaction and correct for sample-to-sample variations in reverse transcriptase PCR efficiency. See ABI TaqMan Human Endogenous Control Plate; TATAA Biocenter Endogenous Control Gene Panel; Ambion: 18S RNA as an Internal Control; Ambion: GAPDH, b-actin, cyclophilin, 18S RNA as internal controls; EXPOLDB: The most constantly expressed housekeeping genes.

Exogenous control: This is a characterized RNA or DNA spiked into each sample at a known concentration. An exogenous active reference is usually an in vitro construct that can be used as an internal positive control (IPC) to distinguish true target negatives from PCR inhibition. An exogenous reference can also be used to normalize for differences in efficiency of sample extraction or complementary DNA (cDNA) synthesis by reverse transcriptase. Whether or not an active reference is used, it is important to use a passive reference dye (usually ROX) in order to normalize for non-PCR-related fluctuations in fluorescence signal.

FAM: 6-carboxy fluorescein. Most commonly used reporter dye at the 5' end of a TaqMan® probe.

Fast PCR: A modified PCR protocol that allows shortening of overall reaction time to less than the typical 90 minutes (usually 40 minutes or less) thanks to recent developments in amplicon design, reagent chemistry, thermocycling conditions as well as the PCR machines with fast ramping rates. See Biocompare Tutorials > Fast PCR (text).

Fluorescence resonance energy transfer (FRET): The interaction between the electronic excited states of two dye molecules. The excitation is transferred from one (the donor) dye molecule to the other (the acceptor) dye molecule. FRET is distance-dependent and occurs when the donor and the acceptor dye are in close proximity.

High resolution melting (HRM) curve analysis: See Melting curve (dissociation) analysis.

Housekeeping gene: Genes that are widely expressed in abundance and are usually used as reference genes for normalization in real-time PCR with the assumption of 'constant expression'. The current trend is first to check which housekeeping genes are suitable for the target cell or tissue and then to use more than one of them in normalization in qPCR assays. See for EXPOLDB: The most constantly expressed housekeeping genes housekeeping genes showing the least inter-individual difference in their expression levels.

Hybridization probe: One of the main fluorescence-monitoring systems for DNA amplification. LightCycler probes are hybridization probes and are not hydrolyzed by Taq Polymerase. For this reason, melting curve analysis is possible with hybridization probes. See Wittwer, 1997 and Hybridization Probe Chemistry for details.

Hydrolysis probe: One of the main fluorescence-monitoring systems for DNA amplification. TaqMan® probes are an example. These kinds of probes are hydrolyzed by the 5' endonuclease activity of Taq Polymerase during PCR. See Wittwer, 1997 for details.

Internal positive control (IPC): An exogenous IPC can be added to a multiplex assay or run on its own to monitor the presence of inhibitors in the template. Most commonly the IPC is added to the PCR master mix to determine whether inhibitory substances are present in the mix. Alternatively, it can be added at the point of specimen collection or prior to nucleic acid extraction to monitor sample stability and extraction efficiency, respectively.

LATE (Linear After The Exponential)-PCR: A new form of asymmetric PCR that uses primer pairs deliberately designed for use at unequal concentrations (Pierce, 2003; Sanchez, 2004). Unlike typical asymmetric PCR, LATE-PCR, amplification is efficient due to improved primer design (Pierce, 2005). LATE-PCR begins with an exponential phase in which amplification efficiency is similar to that of symmetric PCR. Once the limiting primer is depleted, the reaction abruptly switches to linear amplification, and the single-stranded product is made for many additional thermal cycles. LATE-PCR consistently generates strong signals because the absence of product strand reannealing permits unhindered hybridization of the molecular beacon to its target strand and continued accumulation of that strand beyond the cycle at which symmetric reactions typically plateau. By eliminating the exponential phase, LATE-PCR generates less error scatter among replicates. When used in conjunction with molecular beacons, LATE-PCR results in increased signal intensity and reduced sample variation. These features are particularly useful for real-time PCR initiated with single cells. LATE-PCR has been used to directly amplify ssDNA for pyrosequencing (Salk, 2006). See also Bonetta, 2005.

Light-up probe: The light-up probe is a peptide nucleic acid (PNA) oligomer to which an asymmetric cyanine dye thiazole orange (a single reporter dye) is tethered. Upon hybridization the thiazole orange moiety interacts with the nucleic acid bases and the probe becomes brightly (up to 50-fold) fluorescent (Svanvik, 2000a; 2000b & 2001;Isacsson, 2000; Wolffs, 2001). Being based on an uncharged analog (PNA), the light-up probe hybridizes faster and binds target DNA much stronger than oligonucleotide-based probes. See also LightUp Technologies.

Linear View: Amplification plot view displayed using exact DRn values on the Y-axis. The alternative is the log-view, which expands the initiation of exponential amplification phase (and also the baseline period activity). Either can be used for threshold setting.

Locked Nucleic Acid (LNA®) Probes: A new generation of sequence-specific probes designed using LNA (a novel nucleic acid analogue), which has enhanced hybridization performance and biological stability (Koch, 2003; Tolstrup, 2003; Johnson, 2004). LNA has also been used in primers to increase sensitivity (Latorra, 2003). See also web brochures by Proligo; Exiqon; IDT; Gene Link; PCR: Replicating Success (Moore, 2005).

Log-dilution: Serial dilutions in powers of 10 (10, 100, 1000 etc).

Log-view: See Linear View.

LUX^TM (Light Upon eXtension) primers: Created by Invitrogen, LUX^TM primer sets include a self-quenched fluorogenic primer and a corresponding unlabeled primer. The labeled primer has a short sequence tail of 4–6 nucleotides on the 5′ end that is complementary to the 3′ end of the primer. The resulting hairpin secondary structure provides optimal quenching of the fluorophore. When the primer is incorporated into double-stranded DNA during PCR, the fluorophore is dequenched and the signal increases by up to ten-fold. By eliminating the need for a quencher dye, the LUX^TM primers reduce the cost (LUX^TM vs TaqMan^®).

Melting curve (dissociation) analysis: Every piece of dsDNA has a melting point (Tm) at which temperature 50% of the DNA is single stranded. The temperature depends on the length of the DNA, sequence order, G:C content and Watson-Crick pairing. When DNA-binding dyes are used, as the fragment is heated, a sudden decrease in fluorescence is detected when Tm is reached (due to dissociation of DNA strands and release of the dye). This point is determined from the inflection point of the melting curve or the melting peak of the derivative plot (what is meant by derivative plot is the negative first-derivative of the melting curve). The same analysis can be performed when hybridization probes are used as they are still intact after PCR. As hydrolysis probes (e.g., TaqMan®) are cleaved during the PCR reaction, no melting curve analysis possible if they are used (because of their specificity, there is no need either). Mismatch between a hybridization probe and the target results in a lower Tm. Melting curve analysis can be used in known and unknown (new) mutation analysis as a new mutation will create an additional peak or change the peak area. See Ririe, 1997 for details of melting curve analysis. High-resolution melting curve analysis can be achieved on dedicated instruments like Idaho Technology's LightScanner^® or on Corbett’s Rotor-Gene 6000.

Minor groove binders (MGBs): These dsDNA-binding agents are attached to the 3’ end of TaqMan® probes to increase the Tm value (by stabilization of hybridization) and to design shorter probes. Longer probes reduce design flexibility and are less sensitive to mismatch discrimination. MGBs also reduce background fluorescence and increase dynamic range due to increased efficiency of reporter quenching (these probes use non-fluorescent quenchers at the 3’end). By allowing the use of shorter probes with higher Tm values, MGBs enhances mismatch discrimination in genotyping assays. See ABI Allelic Discrimination with TaqMan® Probes.

Minus reverse transcriptase control (^_ RTC): A quantitative real-time PCR control sample that contains the starting RNA and all other components for one-step reaction but no reverse transcriptase. Any amplification suggests genomic DNA contamination.

Molecular beacons: These hairpin probes consist of a sequence-specific loop region flanked by two inverted repeats. Reporter and quencher dyes are attached to each end of the molecule and remain in close contact unless sequence-specific binding occurs and reporter emission (FRET) occurs. See How it Works.

Monte Carlo effect: Problems with reproducible quantification of low abundance targets (<1000 copies) by qPCR. It is a limitation of PCR amplification from small amounts of any complex template due to differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration; the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified product. Originally described by Karrer, 1995; see Bustin & Nolan, 2004 for details.

Multiplexing: Simultaneous analysis of more than one target. Specific quantification of multiple targets that are amplified within a reaction can be performed using a differentially labeled primer or probes. Amplicon or probe melting curve analysis allows multiplexing in allelic discrimination if a dsDNA-binding dye is used as the detection chemistry.

Normalization: A control gene that is expressed at a constant level is used to normalize the gene expression results for variable template amount or template quality. If RNA quantitation can be done accurately, normalization might be done using total RNA amount used in the reaction. The use of multiple housekeeping genes that are most appropriate for the target cell or tissue is the most optimal means for normalization. This normalization is performed by the experimenter and should not be mixed up with the normalization of fluorescence signal using the passive reference dye (usually ROX) performed by the equipment.

Nucleic acid sequence based amplification (NASBA): NASBA is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the coordinated activity of THREE enzymes: AMV reverse transcriptase, RNase H and T7 RNA polymerase. NASBA allows direct detection of viral RNA by nucleic acid amplification. For examples, see Loens, 2003; Guichon, 2004.

No amplification controls (NAC, a minus enzyme control): In mRNA analysis, NAC is a mock reverse transcription containing all the RT-PCR reagents, except the reverse transcriptase. If cDNA or genomic DNA is used as a template, a reaction mixture lacking Taq polymerase can be included in the assay as NAC. No product should be synthesized in the NTC or NAC. 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.

No template controls (NTC, a minus sample control): NTC includes all of the RT-PCR reagents except the RNA template. No product should be synthesized in the NTC or NAC; if a product is amplified, this indicates contamination (fluorescent or PCR products) or presence of genomic DNA in the RNA sample. NTC is not equivalent to H₂O controls and H₂O controls are not used in qPCR experiments.

Normalized amount of target: A unitless number that can be used to compare the relative amount of target in different samples.

Nucleic acid target: (also called “target template”) - DNA or RNA sequence that is going to be amplified.

Passive reference (reference dye): A fluorescence dye that provides an internal reference to which the reporter dye signal can be normalized during data analysis by the software. This type of normalization is necessary to correct for fluctuations from well to well caused by changes in concentration or volume. ROX is the most commonly used passive reference dye.

Peltier element: The element used for heating and cooling in a qPCR machine. Peltier coolers (in ABI machines) use electron flow between semiconductor couples to heat or cool one side of a plate depending on the direction of current. Other systems use liquid or air flow or mechanical transition between blocks of different temperatures to cycle the samples.

Platform: Refers to hardware that performs real-time PCR. For a current list of available machines, see Michael Pfaffl’s page & Biocompare.

PNA (peptide nucleic acid oligomer): See light-up probe.

Primer Express^® Software: A primer design algorithm by ABI. It designs TaqMan^® primer and probe sets to be used at standard conditions of ABI real-time PCR equipment. See Designing TaqMan MGB Probe and Primer Sets for Gene Expression Using Primer Express Software v.2.0 and ABI Taqman Primer/Probe Design using Primer Express.

Quencher: The molecule that absorbs the emission of fluorescent reporter when in close vicinity. Most commonly used quenchers include TAMRA, DABCYL and BHQ. The quenchers are usually at the 3’ end of a dual-labeled fluorescent probe. Quencher dye is also called acceptor.

R: In illustrations of real-time PCR principles, 'R' represents fluorescent Reporter (fluorochrome).

r coefficient: Correlation coefficient, which is used to analyze a standard curve (ten-fold dilutions plotted against C_t values) obtained by linear regression analysis. It should be ≥ 0.99 for gene quantitation analysis. It takes values between zero and -1 for negative correlation and between zero and +1 for positive correlations.

R² coefficient: Usually mixed up with 'r' but this is R-squared (also called coefficient of determination). This coefficient only takes values between zero and +1. R² is used to assess the fit of the standard curve to the data points plotted. The closer the value to 1, the better the fit.

Rapid-cycle PCR: A powerful fast PCR technique for nucleic acid amplification and analysis that is completed in less than half an hour. Samples amplified by rapid-cycle PCR are immediately analyzed by melting curve analysis in the same instrument. In the presence of fluorescent hybridization probes, melting curves provide ‘dynamic dot blots’ for fine sequence analysis, including SNPs. Leading instruments that perform rapid-cycle PCR are RapidCycler2 (Idaho Technology) and LightCycler (Roche).

Real-time PCR: The continuous collection of fluorescent signal from polymerase chain reaction throughout cycles.

Reference: A passive or active signal used to normalize experimental results. Endogenous and exogenous controls are examples of active references. Active reference means the signal is generated as the result of PCR amplification.

Reference dye: Used in all reactions to obtain normalized reporter signal (Rn) adjusted for well-to-well variations by the analysis software. The most common passive reference dye is ROX and is usually included in the master mix. Not all instruments require the use of a reference dye (see Table 1 in Real-Time PCR by Qiagen).

Reporter dye (fluorophore): The fluorescent dye used to monitor amplicon accumulation. This can be attached to a specific probe or can be a dsDNA-binding agent (see for example SYBR^® Green I). For specifications of common reporters, see Table 1 and Figure 1 in Real-Time PCR by Qiagen.

Relative quantitation: A relative quantification assay is used to analyze changes in gene expression in a given sample relative to another reference sample (such as relative increase or decrease -compared to the baseline level- in gene expression in response to a treatment or in time etc). Includes comparative C_t (DDC_t) and relative-fold methods. (Relative Quantification Page by Pfaffl).

Ribosomal RNA (rRNA): Commonly used as a normalizer in quantitative real-time RNA. It is not considered ideal due to its expression levels, transcription by a different RNA polymerase and possible imbalances in relative rRNA-to-mRNA content in different cell types.

Rn (normalized reporter signal): The fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye. Rn+ is the Rn value of a reaction containing all components, including the template and Rn– is the Rn value of an unreacted sample. The Rn– value can be obtained from the early cycles of a real-time PCR run (those cycles prior to a significant increase in fluorescence), or a reaction that does not contain any template.

DRn (delta Rn, dRn): The magnitude of the fluorescence signal generated during the PCR at each time point. The DRn value is determined by the following formula: (Rn+) – (Rn–).

ROX: 6-carboxy-X-rhodamine. Most commonly used passive reference dye for normalization of reporter signal. The emission recorded from ROX during the baseline cycles (usually 3 to 15) is used to normalize the emission recorded from the reporter due to amplification in later cycles. The use of ROX improves the results by compensating for small fluorescent fluctuations such as bubbles and well-to-well variations that may occur in the plate. 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.

Scorpion: A fluorescence detection system consists of a detection probe with the upstream primer with a fluorophore at the 5' end, followed by a complementary stem-loop structure also containing the specific probe sequence, quencher dye and a PCR primer on the 3' end. Between the primer and its tail (the probe), a blocking agent (DNA spacer, hexaethylene glycol) is placed. This structure makes the sequence-specific priming and probing a unimolecular event that creates enough specificity for allelic discrimination assays. See How it Works and Scorpion Technology.

Slope: Mathematically calculated slope of standard curve, e.g., the plot of C_t values against logarithm of ten-fold dilutions of target nucleic acid. This slope is used for efficiency calculation. Ideally, the slope should be –3.3 (–3.1 to –3.6), which corresponds to 100% efficiency (precisely 1.0092) or two-fold (precisely, 2.0092) amplification at each cycle. Also called gradient. See Stratagene Slope to Efficiency Calculator.

Standard: A sample of known concentration used to construct a standard curve. By running standards of varying concentrations, a standard curve is created from which the quantity of an unknown sample can be calculated.

Standard curve: Obtained by plotting C_t values against log-transformed concentrations of serial ten-fold dilutions of the target nucleic acid. Standard curve is obtained for quantitative PCR and the range of concentrations included should cover the expected unknown concentrations range. It is used to find out the dynamic range of the target (and/or normalizer), to calculate the slope (therefore, efficiency), r and R² coefficients and also to help with quantitation.

Sunrise^TM primers: Originally created by Oncor, sunrise^TM primers are similar to molecular beacons. They are self-complementary primers that dissociate through the synthesis of the complementary strand and produce fluorescence signals. See also LUX primers.

SYBR^® Green I: A fluorogenic minor groove binding dye that emits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA. It is used as a cheaper alternative in real-time PCR applications. It does not bind to ssDNA but because of the lack of sequence specificity it binds to any dsDNA product. Its use usually requires melting curve analysis to assure specificity of the results (and if multiplexing is attempted). See Morrison, 1998 and How it Works.

TAMRA: 6-carboxy-terta-methyl-rhodamine. Most commonly used quencher at the 3' end of a TaqMan^® probe.

TaqMan^® probe: A dual-labeled specific hydrolysis probe designed to bind to a target sequence with a fluorescent reporter dye at one end (5’) and a quencher at the other (3’). Assays using Taqman probes are also called 5' nuclease assays. See How it Works.

Threshold: Usually 10X the standard deviation of Rn for the early PCR cycles (baseline). The threshold should be set in the region associated with an exponential growth of PCR product (which may be easier is the log-view of the amplification plot is used). It is assigned for each run to calculate the C_t value for each amplification.

Unknown: A sample containing an unknown quantity of template. This is the sample of interest (experimental sample as opposed to positive controls or standards) whose quantity is being determined.

M.Tevfik Dorak, MD PhD

Last updated on 11 June 2007

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