Glossary
217
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
baseline-corrected
normalized reporter
(
∆
Rn)
The magnitude of normalized fluorescence generated by the reporter:
1.
In experiments that contain data from real-time PCR, the magnitude of normalized
fluorescence generated by the reporter at each cycle during the PCR amplification. In
the
∆
Rn vs. Cycle amplification plot,
∆
Rn is calculated at each cycle as:
∆
Rn (cycle) = Rn (cycle)
−
Rn (baseline), where Rn = normalized reporter
2.
In genotyping experiments and presence/absence experiments, the difference in
normalized fluorescence generated by the reporter between the pre-PCR read and the
post-PCR read. In the allelic discrimination plot (genotyping experiments) and the
presence/absence plot (presence/absence experiments),
∆
Rn is calculated as:
∆
Rn = Rn (post-PCR read)
−
Rn (pre-PCR read), where Rn = normalized reporter
See also normalized reporter (Rn).
biological replicates
Reactions that contain identical components and volumes, but evaluate separate samples of
the same biological source (for example, samples from three different mice of the same
strain, or separate extractions of the same cell line or tissue sample). See also technical
replicates.
blocked IPC
In presence/absence experiments, a reaction that contains IPC blocking agent, which blocks
amplification of the internal positive control (IPC). In the 7500 software, the task for the
IPC target in wells that contain IPC blocking agent. See also negative control-blocked IPC
wells.
C
T
See threshold cycle (C
T
).
calibrator
See reference sample.
chemistry
See reagents.
comparative
C
T
(
∆∆
C
T
) method
Method for determining relative target quantity in samples. With the comparative C
T
(
∆∆
C
T
)
method, the 7500 software measures amplification of the target and of the endogenous
control in samples and in a reference sample. Measurements are normalized using the
endogenous control. The software determines the relative quantity of target in each sample
by comparing normalized target quantity in each sample to normalized target quantity in the
reference sample.
custom dye
Dye that is not supplied by Applied Biosystems. Custom dyes may be adapted for use in
experiments on the 7500/7500 Fast system. When using custom dyes, the custom dye
should be added to the Dye Library and a custom dye calibration performed.
cycle threshold
See threshold cycle (C
T
).
cycling stage
In the thermal profile, a stage that is repeated. A cycling stage is also called an amplification
stage. For cycling stages, you can enable AutoDelta settings. See also amplification stage.
