Glossary

This glossary has been adapted from the Editor's Real-Time PCR webpage (http:// www.dorak.info/genetics/realtime.html). The online version includes references and hyperlinks.

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).

Allelic discrimination assay: Assays designed to type for gene variants. Either differentially labeled probes (one for each variant) or a single probe and melting curve analysis can be used for this purpose. Alternatively, dsDNA-binding dyes can be used in combination with melting curve analysis.

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® probes that hybridize on the target sequence in close proximity. The anchor probe (donor) emits the fluorescein to excite the reporter probe (acceptor) to initiate fluorescence resonance energy (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).

Coefficient of variance (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 Ct 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.

Ct (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 Ct 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 (Cp) 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°C 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 Ct values are obtained. If endogenous control is used for AACt 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 = l0(-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 Ct vs logtemplate 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 ampli-con 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.

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.

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-6-carboxy-X-rhodamine) 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.

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.

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.

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.

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' endonucle-ase activity of Taq Polymerase during PCR.

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.

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

LUX™ (Light upon extension) primers: Created by Invitrogen, LUX™ primer sets include a self-quenched fluorogenic primer and a corresponding unlabeled primer. The labeled primer has a short sequence tail of 46 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™ primers reduce the cost.

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. 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.

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. By allowing the use of shorter probes with higher Tm values, MGBs enhances mismatch discrimination in genotyping assays.

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.

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.

Nucleic acid sequence based amplification (NASBA): NASBA is an isothermal nucleic acid amplification procedure based on target-specific primers and probes, and the co-ordinated activity of THREE enzymes: AMV reverse transcriptase, RNase H and T7 RNA polymerase. NASBA allows direct detection of viral RNA by nucleic acid amplification.

No amplification control (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 control (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.

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: A dye that provides an internal reference to which the reporter dye signal can be normalized during data analysis. 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.

Quencher: The molecule that absorbs the emission of fluorescent reporter when in close vicinity. Most commonly used quenchers include 6-carboxy-tetra-methyl-rhodamine (TAMRA), (DABCYL) and black hole quencher (BHQ).

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 Ct 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: Frequently 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 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 single nucleotide polymorphism (SNPs). Leading instruments that perform rapid-cycle PCR are RapidCycler®2 (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 reference dye is ROX and is usually included in the master mix.

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.

Relative quantification: 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 Ct (AACt) and relative-fold methods.

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.

ARn (delta Rn, dRn): The magnitude of the signal generated during the PCR at each time point. The ARn 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.

Scorpion: Another 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. This structure makes the sequence-specific priming and probing a unimolecular event which creates enough specificity for allelic discrimination assays.

Slope: Mathematically calculated slope of standard curve, e.g., the plot of Ct 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.

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 Ct 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.

Sunrise™ primers: Created by Oncor, Sunrise™ primers are similar to molecular beacons. They are self-complementary primers which dissociate through the synthesis of the complementary strand and produce fluorescence signals.

TAMRA (6-carboxy-tetra-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 and a quencher at the other.

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. It is the numerical value assigned for each run to calculate the Ct value for each amplification.

Unknown: A sample containing an unknown quantity of template. This is the sample of interest whose quantity is being determined.

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