Terms Used in Quantitation Analysis
Active reference - Active reference means the signal is generated as the result of PCR amplification. The active reference has its own set of primers and probe.
Endogenous and exogenous controls are examples of active references.
Amplicon A short segment of DNA generated by the PCR process.
Amplification plot The plot of fluorescence signal versus cycle number.
Baseline The initial cycles of PCR, in which there is little change in fluorescence signal.
Calibrator A sample used as the basis for comparative results.
CT (threshold cycle) The fractional cycle number at which the fluorescence passes the fixed threshold
Endogenous control – This is an RNA or DNA that is present in each experimental sample as isolated. By using an endogenous control as an active reference, you can normalize quantitation of a messenger RNA (mRNA) target for differences in the amount of total RNA added to each reaction.
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 containing the dye ROX in order to normalize for non-PCR-related fluctuations in fluorescence signal.
Normalized amount of target - A unitless number that can be used to compare the relative amount of target in different samples.
NTC (no template control) - A sample that does not contain template. It is used to verify amplification quality.
Nucleic acid target (also called “target template”) - DNA or RNA sequence that you wish to amplify
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 forestallment fluctuations caused by changes in concentration or volume.
Reference A passive or active signal used to normalize experimental results.
Rn (normalized reporter) - The fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye
Rn+ The Rn value of a reaction containing all components, including the template
Rn- The Rn value of an un-reacted sample. The Rn- value can be obtained from:
- The early cycles of a real-time PCR run (those cycles prior to a detectable increase in fluorescence), OR
- A reaction that does not contain any template
ΔRn (delta Rn) - The magnitude of the signal generated by the given set of PCR conditions. The Δ Rn value is determined by the following formula: (Rn+) – (Rn-)
Standard A sample of known concentration used to construct a standard curve. By running standards of varying concentrations, you create a standard curve from which you can extrapolate the quantity of an unknown sample.
Threshold The average standard deviation of Rn for the early PCR cycles, multiplied by an adjustable factor. The threshold should be set in the region associated with an exponential growth of PCR product.
Unknown A sample containing an unknown quantity of template. This is the sample whose quantity you want to determine.
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