Triple primer polymerase chain reaction. A new way to quantify truncated mRNA expression.
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.
Leygue
E
E
Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Canada.
Murphy
L
L
Kuttenn
F
F
Watson
P
P
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
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011960:Receptors, Estrogen