Quantitative reverse transcriptase PCR

Overview

RT-RT-PCR or Real Time Reverse Transcriptase PCR is a method to quantify the amount of mRNA for a gene of interest present in a pool of isolated RNA. It is generally more precise than traditional Reverse Transcription PCR (RT-PCR) because the generation of product is continously monitored during the PCR run (this is where the term Real Time comes in), rather than at the end of a PCR reaction (traditional or "endpoint" PCR).

Generation of gene product is detected using fluorescent markers such as the DNA binding dye SYBR Green. The intensity of fluorescence directly correlates with a quantity of DNA present in a reaction.

The precision afforded by RT-RT-PCR comes about because it is possible to identify the cycle during which the fluorescence reaches an arbitrary threshold, idealing set during the logarithmic amplification phase of DNA amplification. During the log phase each copy of DNA is being amplified, and thus can be a better measure than endpoint PCR, where reagents such a nucleotides may become exhausted and result in inefficient amplification, resulting in inaccurate quantitation of the gene of interest.

Materials

Roche Light Cycler

LightCycler FastStart DNA MasterPLUS SYBR Green I

RNA

Procedure

Endy:Real-time RT-PCR

See also

• PCR techniques