Applied Biosystems (ABI)

RT-PCR

Real-time reverse transcription (RT) polymerase chain reaction (PCR) is the most sensitive and reliable method for the detection and quantitation of nucleic acids levels. The Affymetrix Resource Facility has acquired the ABI 7900 sequence detection system which allows real time detection of PCR products as they accumulate during PCR and thus enables the accurate quantitation of DNA and RNA over a wide dynamic range. The ABI 7900 instrument can be used for various applications such as quantitation of gene expression, allelic discrimination (SNP detection) and pathogen detection.

The delta-delta Ct method of RT-PCR is ideal for primer pairs with an efficiency of 90% or greater AND for genes that will show a large fold change in gene expression (5-fold or greater).  The standard curve method will control for differences in primer efficiency.  If the fold change data from a standard curve run is in agreement with delta-delta Ct-calculated fold changes, then subsequent plates of RT-PCR using the same primer pairs can be run using the delta-delta Ct method.

Quantitative Real-Time PCR Reference Materials:

Real-Time Quantitative PCR Overview

Designing Probes and Primer by using Primer Express

Assay Design and Optimization

Relative Quantitation of Gene Expression