Chapter 2
Design the Relative Standard Curve Experiment
Define the Methods and Materials
25
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 relative standard curve experiment, you:
• Select
Relative Standard Curve
as the quantitation method. The relative standard
curve method is used to determine relative target quantity in samples. When setting
up your reaction plate, the relative standard curve method requires targets,
standards, samples, a reference sample, and an endogenous control.
• 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.
• Select the appropriate ramp speed for the instrument run:
IMPORTANT!
Select a Fast run only if you use a 7500 Fast system. Fast runs cannot
be performed on a 7500 system.
– 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.
– Select
RNA
if you are performing 1-step RT-PCR. You are adding total RNA or
mRNA to the PCR reactions.
IMPORTANT!
To use the Fast ramp speed with RNA templates, you must
design your experiment using Advanced Setup instead of the Design Wizard.
– Select
gDNA (genomic DNA)
if you have already extracted the gDNA from
tissue or sample. You are adding purified genomic DNA to the PCR reactions.
