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TaqMan™ Fluorogenic Detection System to Analyze Gene Transcription in Autopsy Material

  • Kaori Shintani-Ishida
  • Bao-Li Zhu
  • Hitoshi Maeda
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1105)

Abstract

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 words

Real-time PCR TaqMan fluorogenic detection system Quantification mRNA Autopsy material TaqMan probe 

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Copyright information

© Humana Press 2014

Authors and Affiliations

  • Kaori Shintani-Ishida
    • 1
  • Bao-Li Zhu
    • 2
  • Hitoshi Maeda
    • 2
  1. 1.Department of Legal MedicineOsaka City University Medical SchoolOsakaJapan
  2. 2.Department of Legal MedicineOsaka City University Medical SchoolOsakaJapan

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