Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium Protocol uses a single thermostable RNA polymerase to perform high-specificity RT-PCR. A high-temperature RT reaction is followed by PCR amplification of the cDNA using a single thermostable poymerase, the GeneAmp AccRT RNA PCR enzyme from Applied Biosystems. The high temperature of the RT reaction enhances the specificity of primer binding and also reduces secondary structure in the template, thereby increasing the efficiency of polymerization.
Competitive RT-PCR: Estimation of Copy Number Protocol In this protocol, sample and competitor RNAs are reverse transcribed (separately) in a pilot experiment. A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. Procedure provides an approximate copy number for the sample, which is then fine-tuned by repeating the experiment with a series of twofold dilutions of competitor. The experiment includes controls for sample-to-sample variations in RT efficiency.
Competitive RT-PCR: Preparation of Competitor RNA Protocol The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor. This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest. Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out.
Real-Time RT-PCR: cDNA Synthesis Protocol Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA. RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue.
Reverse Transcriptase In Situ PCR Protocol Protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in Rnase-free Dnase that is performed after protease digestion, and an RT step, prior to in situ PCR.