Chapter 7 Prepare the Comparative C
T
Reactions
Prepare the Template
139
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative C
T
Experiments
Notes
Prepare the Template
Prepare the template for the PCR reactions using the High-Capacity cDNA Reverse
Transcription Kit.
IMPORTANT! Applied Biosystems recommends that you use the High-Capacity cDNA
Reverse Transcription Kit to reverse-transcribe cDNA from total RNA. The TaqMan
®
Gene Expression Assays are designed to be compatible with the High-Capacity cDNA
Reverse Transcription Kit; other protocols have not been tested for use with the TaqMan
Gene Expression Assays.
About the
Example
Experiment
For the comparative C
T
example experiment, the template for the PCR reactions is cDNA
reverse-transcribed from total RNA samples using the High-Capacity cDNA Reverse
Transcription Kit.
Required
Materials
• One of the following Ambion
®
starter packs for RNA isolation:
Kit Contents
Catalog
Number
qRT-PCR
Starter Pack
RNAlater
®
Tissue Collection: RNA Stabilization
Solution
AM7020
RNaseZap
®
Wipes AM9786
RT-PCR Grade Water (nuclease-free) AM9935
Silencer
®
Validated siRNA, Std Purity AM51331
Choice of RNA sample preparation products:
•RNAqueous
®
-4PCR Kit
Note: Recommended if you are isolating RNA from
cells or tissues that may be difficult to disrupt or
have high RNase activity.
AM1914
•RiboPure
™
Kit
Note: Recommended if you are:
• Isolating RNA from all tissues, including those
that may be difficult to disrupt, are rich in lipids,
or have high RNase activity, or
• Labeling and amplifying RNA for use on
microarrays.
AM1924
•TURBO DNA-free
™
Note: Recommended if you are using SYBR
®
Green reagents. If you are using SYBR Green
reagents, use TURBO DNA-free in conjunction with
the RiboPure Kit.
AM1907
To Next Page