This Article Order Full text via Infotrieve Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Leygue, E. Articles by Watson, P. Search for Related Content PubMed PubMed Citation Articles by Leygue, E. Articles by Watson, P.

American Journal of Pathology, Vol 148, 1097-1103, Copyright © 1996 by American Society for Investigative Pathology

REGULAR ARTICLES

Triple primer polymerase chain reaction. A new way to quantify truncated mRNA expression

E Leygue, L Murphy, F Kuttenn and P Watson
Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Canada.

The most practical method to quantify mRNA expression within small tumor samples is reverse transcription (RT) followed by quantitative polymerase chain reaction (PCR). One approach, known as "competitive RT- PCR" allows absolute quantitation by reference to synthetic RNA standards but is time-consuming and requires multiple manipulations that limit its usefulness as a screening assay. We describe here a new approach to quantify truncated type mRNAs relative to the wild-type transcripts in small amounts of tissue. This technique, called RT- triple primer-PCR, consists of coamplification of wild-type and truncated cDNAs using three primers in the PCR. To validate this approach, a truncated estrogen receptor variant (clone 4) was quantified relative to the wild-type estrogen receptor using plasmid preparations. The ratio of triple primer-PCR products obtained was directly related to the initial ratio of input cDNAs. RT-triple primer- PCR was then used to compare the relative expression of clone 4 mRNA in frozen sections of normal human breast tissue and human breast tumors with characteristics of good prognosis. The statistically significant difference (P = 0.03) observed between normal and tumor tissues suggests that elevated expression of the clone 4 variant may be associated with early steps of tumorigenesis. This technique provides a useful alternative to already described quantitative RT-PCR techniques for the quantification of truncated mRNA within small amounts of biological material.

This article has been cited by other articles:

				O.N. Richter, K. Kubler, J. Schmolling, M. Kupka, J. Reinsberg, U. Ulrich, H. van derVen, E. Wardelmann, and K. van derVen Oxytocin receptor gene expression of estrogen-stimulated human myometrium in extracorporeally perfused non-pregnant uteri Mol. Hum. Reprod., May 1, 2004; 10(5): 339 - 346. [Abstract] [Full Text] [PDF]

				N. Wedemeyer, T. Potter, S. Wetzlich, and W. Gohde Flow Cytometric Quantification of Competitive Reverse Transcription-PCR Products Clin. Chem., September 1, 2002; 48(9): 1398 - 1405. [Abstract] [Full Text] [PDF]

				L. C. Murphy, S. L. R. Simon, A. Parkes, E. Leygue, H. Dotzlaw, L. Snell, S. Troup, A. Adeyinka, and P. H. Watson Altered Expression of Estrogen Receptor Coregulators during Human Breast Tumorigenesis Cancer Res., November 1, 2000; 60(22): 6266 - 6271. [Abstract] [Full Text]

				G. Castaldo, G. Calcagno, R. Sibillo, R. Cuomo, G. Nardone, L. Castellano, C. Del Vecchio Blanco, G. Budillon, and F. Salvatore Quantitative Analysis of Aldolase A mRNA in Liver Discriminates between Hepatocellular Carcinoma and Cirrhosis Clin. Chem., July 1, 2000; 46(7): 901 - 906. [Abstract] [Full Text] [PDF]

				E. Leygue, H. Dotzlaw, P. H. Watson, and L. C. Murphy Expression of the Steroid Receptor RNA Activator in Human Breast Tumors Cancer Res., September 1, 1999; 59(17): 4190 - 4193. [Abstract] [Full Text] [PDF]

				E. Leygue, H. Dotzlaw, P. H. Watson, and L. C. Murphy Expression of Estrogen Receptor {beta}1, {beta}2, and {beta}5 Messenger RNAs in Human Breast Tissue Cancer Res., March 1, 1999; 59(6): 1175 - 1179. [Abstract] [Full Text] [PDF]