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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Alwine, J. C, Kemp, D. J., and Stark, G. R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350–5354.
Hod, Y. (1992) A simplified ribonuclease protection assay. BioTechniques 13, 852–854.
Saccomanno, C. F., Bordonaro, M., Chen, J. S., and Nordstrom, J. L. (1992) A faster ribonuclease protection assay. BioTechniques 13, 846–850.
Boorstein, W. R. and Craig, E. A. (1989) Primer extension analysis of RNA. Methods Enzymol. 180, 347–369.
Freeman, W. M., Walker, S. J., and Vrana, K. E. (1999) Quantitative RT-PCR: pitfalls and potential. BioTechniques 26, 112–122.
Becker-Andre, M. and Hahlbrock, K. (1989) Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res. 17, 9437–9446.
Wiesner, R. J. (1992) Direct quantification of picomolar concentrations of mRNAs by mathematical analysis of a reverse transcription/exponential polymerase chain reaction assay. Nucleic Acids Res. 20, 5863–5864.
Gibson, U. E., Heid, C. A., and Williams, P. M. (1996) A novel method for real time quantitative RT-PCR. Genome Res. 6, 995–1001.
Ishida, K., Zhu, B. L., and Maeda, H. (2000) Novel approach to quantitative reverse transcription PCR assay of mRNA component in autopsy material using the TaqMan fluorogenic detection system: dynamics of pulmonary surfactant apoprotein A. Forensic Sci. Int. 113, 127–131.
Ishida, K., Zhu, B. L., and Maeda, H. (2002) A quantitative RT-PCR assay of surfactant-associated protein A1 and A2 mRNA transcripts as a diagnostic tool for acute asphyxial death. Legal Med. 4, 7–12.
Kuimelis, R. G., Livak, K. J., Mullah, B., and Andrus, A. (1997) Structural analogues of TaqMan probes for real-time quantitative PCR. Nucleic Acids Symp. Serv. 37, 255–256.
Longley, M. J., Bennett, S. E., and Mosbaugh, D. W. (1990) Characterization of the 5′ to 3′ exonuclease associated with Thermus aquaticus DNA polymerase. Nucleic Acids Res. 18, 7317–7322.
Ercolani, L., Florence, B., Denaro, M., and Alexander, M. (1988) Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. J. Biol. Chem. 263, 15335–15341.
Nakajima-Iijima, S., Hamada, H., Reddy, P., and Kakunaga, T. (1985) Molecular structure of the human cytoplasmic β-actin gene: interspecies homology of sequences in the introns. Proc. Natl. Acad. Sci. USA 82, 6133–6137.
Torczynski, R. M., Fuke, M., and Bollon, A. P. (1985) Cloning and sequencing of a human 18s ribosomal RNA gene. DNA 4, 283–291.
Relative quantification of gene expression (1997) ABI PRISM 7700 Sequence Detection System User Bulletin #2, Perkin Elmer, Foster City, CA, pp. 11–13.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Shintani-Ishida, K., Zhu, BL., Maeda, H. (2005). TaqMan® Fluorogenic Detection System to Analyze Gene Transcription in Autopsy Material. In: Keohavong, P., Grant, S.G. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology™, vol 291. Humana Press. https://doi.org/10.1385/1-59259-840-4:415
Download citation
DOI: https://doi.org/10.1385/1-59259-840-4:415
Publisher Name: Humana Press
Print ISBN: 978-1-58829-084-7
Online ISBN: 978-1-59259-840-3
eBook Packages: Springer Protocols