Quantitative Real-time RT-PCR: Application to Carcinogenesis

FARID E. AHMED

Abstract

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) has simplified and enhanced the quantification of gene expression. However, since no agreed standardizations are available, care must be exercised when designing experiments, including the choice of appropriate amplification primers, detection chemistry and the normalization procedure, in order to obtain meaningful results. Coupling quantitative polymerase chain reaction (qPCR) to cell purification from tumor tissue has made it possible to decrease the variability in expression from in vivo heterogeneous cell populations. Sensitive and specific qRT-PCR has advanced the diagnosis, prognosis and prediction response of colorectal cancer to therapy.

Footnotes

Abbreviations: bp, base pair; CCD, charged coupled device; CRC, colorectal cancer; cDNA, copy deoxyribonucleic acid; DABCYL, 4-4′-dimethylaminophenylazol benzoic acid; DPI₃, dihydrocyclo-pyrroloindole tripeptide; DHPLC, denaturing high performance liquid chromatography; ds, double-stranded; FAM, 6-carboxyfluorescein; HEX, hexachloro-6-carboxyfluorescein; JOE, 2,7,-dimethoxy-4,5-dichloro-6-carboxyfluorescein; LCM, laser capture microdissection; mRNA, messenger ribonucleic acid; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcription polymerase chain reaction; ss, single-stranded; TAMRA, 6-carboxy-N,N,N',N'-tetramethylrhodamine; TET, tetrachloro-6-carboxyfluorescein.
- Received September 28, 2005.
- Accepted October 14, 2005.
Copyright © 2005 The Author(s). Published by the International Institute of Anticancer Research.