TaqMan™ Fluorogenic Detection System to Analyze Gene Transcription in Autopsy Material
Real-time polymerase chain reaction 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 reverse transcription polymerase chain reaction 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.
Key wordsReal-time PCR TaqMan fluorogenic detection system Quantification mRNA Autopsy material TaqMan probe
- 11.Kuimelis RG, Livak KJ, Mullah B, Andrus A (1997) Structural analogues of TaqMan probes for real-time quantitative PCR. Nucleic Acids Symp 37:255–256Google Scholar
- 16.Relative quantification of gene expression (1997) ABI PRISM 7700 sequence detection system user bulletin #2. Perkin-Elmer, Foster City, CA, pp 11–13Google Scholar