title: Triple primer polymerase chain reaction. A new way to quantify truncated mRNA expression.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Leygue
authors.forename: L
authors.initials: L
authors.lastname: Murphy
authors.forename: F
authors.initials: F
authors.lastname: Kuttenn
authors.forename: P
authors.initials: P
authors.lastname: Watson
mesh_terms: D001483:Base Sequence; D001940:Breast / Q000737:chemistry / Q000473:pathology; D001941:Breast Diseases / Q000235:genetics* / Q000473:pathology*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000302:isolation & purification; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis*; D011960:Receptors, Estrogen / Q000235:genetics*; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011960:Receptors, Estrogen