Chapter 1
Get Started
About the 7500/7500 Fast System
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
2
Notes
About the 7500/7500 Fast System
The Applied Biosystems 7500/7500 Fast Real-Time PCR System is a 96-well, five-color
platform that uses fluorescence-based polymerase chain reaction (PCR) reagents to provide:
• Quantitative detection of nucleic acid sequences using real-time analysis.
• Qualitative detection of nucleic acid sequences using end-point and dissociation-
curve analysis.
The Applied Biosystems 7500 Fast Real-Time PCR System enables you to perform
high-speed thermal cycling with run times for quantitative real-time PCR applications
(such as relative quantitation) of fewer than 40 minutes.
About Data
Collection
The 7500/7500 Fast system collects raw fluorescence data at different points during a
PCR, depending on the type of run that the instrument performs:
Regardless of the run type, a data collection point or
read
on the 7500/7500 Fast
instrument consists of three phases:
1.
Excitation
– The instrument illuminates all wells of the reaction plate within the
instrument, exciting the fluorophores in each reaction.
2.
Emission
– The instrument optics collect the residual fluorescence emitted from the
wells of the reaction plate. The resulting image collected by the device consists only
of light that corresponds to the range of emission wavelengths.
3.
Collection
– The instrument assembles a digital representation of the residual
fluorescence collected over a fixed time interval. The 7500 software stores the raw
fluorescence image for analysis.
After a run, the 7500 software uses region of interest (ROI), optical, dye, and background
calibrations to determine the location and intensity of the fluorescence in each read, the
dye associated with each fluorescent signal, and the significance of the signals.
Run Type
Data Collection Point
Real-time
runs
Standard curve
The instrument collects data after each extension step of
the PCR.
Relative standard
curve
Comparative C
T
(
∆∆
C
T
)
Post-PCR
(endpoint)
runs
Genotyping
The instrument collects data:
• Before the PCR. (For presence/absence experiments,
data collection before the PCR is optional but
recommended.)
• (Optional) During the PCR. The instrument can collect
data during the run (real-time); collecting data during
the run can be helpful for troubleshooting.
• After the PCR.
Presence/absence
