Quantitative measurement of mRNAs by polymerase chain reaction

Abstract

Although polymerase chain reaction (PCR) has been used to detect the presence of specific mRNA species, there are no reports indicating that PCR can be used as a reliable, reproducible assay to quantify the relative level of an mRNA. In this study we examined the enzymatic steps (reverse transcription and PCR) required to analyze RNA by PCR and determined the conditions under which the product obtained reproducibly reflects the relative amounts of amplified species in the starting material. Aliquots of total RNA from rat ovaries and GH₃ pituitary cells were used to prepare cDNAs for PCR amplification of ß-actin and prolactin (PRL) sequences, respectively. Assay of equivalent dilutions of ovarian cDNAs made from 10, 2, and 0.4 μg of RNA demonstrated that the amount of PCR product obtained was proportional to both the amount of cDNA amplified and the amount of RNA transcribed, with a relatively small variability for both reactions. cDNAs were also made against RNA prepared from GH₃ cells cultured in the presence or absence of Ca²⁺, which induces PRL gene expression. Measurement of PRL mRNA by PCR gave results comparable to those obtained by Northern blot (4.7-fold induction vs. 5.9-fold), and again was highly reproducible. Additionally, PCR analysis of cDNA against GH₃ nuclear RNA allowed us to detect an apparent splice variant of the PRL nuclear RNA that is also Ca²⁺ regulated. These results indicate the sensitivity and reliability of PCR as a quantitative assay for specific mRNAs, and demonstrate the possibilities for obtaining data not readily available by other means.