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6.2
Real-time PCR Glossary
Absolute quantification
A standard curve is used to quantify unknown samples by
interpolating their quantity.
Allelic Discrimination
Technique used in genotyping to distinguish the presence or
absence of two alleles using two different dye-labelled probes.
Amplicon
Segment of DNA generated by PCR.
Amplification plot
Graphical representation of fluorescence signal versus cycle
number.
Background fluorescence
Fluorescence present in an assay before amplification which is due
to inefficient quenching of the reporter dye, the inherent
fluorescence of the reagents or consumables.
Baseline
The initial cycles of PCR in which there is little change in the
fluorescence.
Calibrator
A specified cDNA or DNA sample to which all other samples in the
experiment will be compared.
cDNA
Complementary DNA produced by reverse transcription of total or
messenger RNA.
Cq
Quantitation cycle, the cycle number at which the fluorescence
increases past a fixed threshold. PrimeQ has default values of 10
standard deviations above the noise for the threshold or Crossing
Line.
Crossing Line
The best fit or threshold, significantly above the noise where the
reaction efficiency is at its highest and most constant for each
amplification plot.
Dissociation Curve
Graphical plot of fluorescence vs. increasing temperature. It is
used for intercalating dyes to determine the temperature at which
the double-stranded DNA is 50% separated.
Endogenous Control
An RNA or DNA template which is present in each sample and is
amplified during the PCR via a second set of primers and probe
e.g. Reference genes.
Exogenous Control
A characterized template which is spiked into each sample at a
known concentration and is amplified during the PCR via a second
set of primers and probe
IPC
Internal Positive Control: A control assay run together with the test
samples as a multiplex. Often used in plus/minus scoring assays to
ensure that a failed PCR is not mistaken for a negative result.
NTC
No template control: Sample which contains all the PCR reaction
components except the DNA template.
