Chapter 6
Design the Comparative C
T
Experiment
Define the Methods and Materials
117
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
Design
Guidelines
When you design your own comparative C
T
experiment:
• Select
Comparative C
T
(
∆∆
C
T
)
as the quantitation method. The comparative C
T
(
∆∆
C
T
) method is used to determine the relative target quantity in samples. When
setting up your reaction plate, the comparative C
T
method requires targets, samples,
a reference sample, and an endogenous control.
Note:
Before you use the comparative C
T
method, Applied Biosystems
recommends that you determine that the PCR efficiencies for the target assay and
the endogenous control assay are approximately equal. Applied Biosystems
TaqMan
®
Gene Expression Assays and Custom TaqMan
®
Gene Expression Assays
have equivalent amplification efficiencies of 100% (
±
10%).
• Select the reagents you want to use:
– Select
TaqMan
®
Reagents
if you want to use TaqMan reagents to detect
amplification and quantify the amount of target in the samples. TaqMan reagents
consist of two primers and a TaqMan probe. The primers are designed to amplify
the target. The TaqMan probe is designed to hybridize to the target and fluoresce
when the target is amplified.
– Select
SYBR
®
Green Reagents
if you want to use SYBR Green reagents to
detect amplification and quantify the amount of target in the samples. SYBR
Green reagents consist of two primers and SYBR Green dye. The primers are
designed to amplify the target. The SYBR Green dye fluoresces when it binds to
double-stranded DNA. SYBR Green dye is often part of the SYBR Green master
mix added to the reaction. If you use SYBR Green dye, select the
Include Melt
Curve
check box to perform melt curve analysis of the amplified target.
IMPORTANT!
Although you can use other fluorescence-based reagents on the
7500/7500 Fast system, you must design your experiment using Advanced Setup
instead of the Design Wizard.
Note:
Fast runs can be performed only on the 7500 Fast system and an upgraded
7500 system.
• Select the appropriate ramp speed for the instrument run:
– Select
Fast (~ 40 minutes to complete a run)
if you are using Fast reagents for
the PCR reactions.
– Select
Standard (~ 2 hours to complete a run)
if you are using standard
reagents for the PCR reactions.
• Select the appropriate PCR template:
– Select
cDNA (complementary DNA)
if you are performing 2-step RT-PCR, and
you have already performed reverse transcription to convert the RNA to cDNA.
You are adding complementary DNA to the PCR reactions.
