QUANTITATIVE-DETERMINATION OF MESSENGER-RNA PHENOTYPES BY THE POLYMERASE CHAIN-REACTION

A method for quantitative determination of specific cellular mRNA is described. The mRNA in a dilution series of total RNA was reverse transcribed by an oligodT primer and the cDNA was amplified by the polymerase chain reaction (PCR) using sets of specific primers. A 32P-or biotin-labeled specific probe was hybridized to the PCR products immobilized on nitrocellulose membrane. The intensity of the hybridization signals was evaluated for quantification of the PCR products. A standard curve was produced by the known amount of the in vitro transcribed cRNA, which contained the same sequence as the mRNA. The series of standard cRNA dilutions were reverse transcribed, amplified and hybridized in the same manner. The amount of the specific RNA was deduced by fitting to the standard curve. Two tissue specimens of intestinal tumors, evaluated on the basis of hybridization signals by three different methods, were shown to contain similar amounts of β-actin mRNA. Furthermore, a Chinese hamster ovary (CHO) cell line transfected with platelet-derived growth factor (PDGF) β-receptor cDNA was found to contain similar amounts of β-actin mRNA as the untransfected CHO cell line. However, the transfected CHO cell line contained over 1011 copies of the PDGF β-receptor mRNA per microgram of total RNA, while the untransfected one showed no detectable RNA, indicating that the latter contained less than 106 copies per microgram of total RNA in this assay. © 1991.