TaqMan fluorogenic detection system to analyze gene transcription in autopsy material.

Abstract

Real-time RT-PCR using a TaqMan fluorogenic detection system is a simple and sensitive assay for quantitative analysis of gene transcription. This method is of potential usefulness in quantifying mRNA of a target gene in autopsy material that has undergone only a small amount of postmortem degradation. The TaqMan fluorogenic detection system can monitor PCR in real time using a dual-labeled fluorogenic hybridization probe (TaqMan probe) and a polymerase with 5'-3' exonuclease activity. The procedures of the quantitative RT-PCR are as follows: RNA is extracted from autopsy material and used to synthesize cDNA by an RT reaction, and the target of interest is amplified and detected by the real-time PCR. The absolute amount of target mRNA in the sample is then determined relative to a standard curve. This chapter describes the methodology of the TaqMan fluorogenic detection system in handling autopsy material in the gene transcription assay.