Glossary
227
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
template
In the Design Wizard of the 7500 software (and in QuickStart for quantitation experiments),
the type of nucleic acid to add to the PCR reaction. The recommended template varies
according to experiment type:
• Quantitation experiments (standard curve, relative standard curve, and comparative
C
T
) – cDNA (complementary cDNA), RNA, or gDNA (genomic DNA)
For quantitation experiments, the template type selection affects the run method,
reaction setup, and materials list.
• Genotyping experiments – Wet DNA (gDNA or cDNA) or dry DNA (gDNA or
cDNA)
For genotyping experiments, the template type selection affects the reaction setup.
• Presence/absence experiments - DNA
For presence/absence experiments, Applied Biosystems recommends adding DNA
templates to the PCR reactions.
thermal profile
Part of the run method that specifies the temperature, time, ramp, and data collection points
for all steps and stages of the 7500/7500 Fast instrument run.
threshold
1.
In amplification plots, the level of fluorescence above the baseline and within the
exponential growth region The threshold can be determined automatically (see
automatic C
T
) or can be set manually (see manual C
T
).
2.
In presence/absence experiments, the level of fluorescence above which the 7500
software assigns a presence call.
threshold cycle (
C
T
)
The PCR cycle number at which the fluorescence meets the threshold in the amplification
plot.
Tm
See melting temperature (Tm).
unknown
In the 7500 software, the task for the target or SNP assay in wells that contain the sample
you are testing:
• In quantitation experiments, the task for the target in wells that contain a sample with
unknown target quantities.
• In genotyping experiments, the task for the SNP assay in wells that contain a sample
with an unknown genotype.
• In presence/absence experiments, the task for the target in wells that contain a sample
in which the presence of the target is not known.
unknown-IPC wells
In presence/absence experiments, wells that contain a sample and internal positive control
(IPC).
y-intercept
In the standard curve, the value of y where the regression line crosses the y-axis. The
y-intercept indicates the expected threshold cycle (C
T
) for a sample with quantity equal to 1.
