Quantitative measurement of mRNAs by polymerase chain reaction.
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 beta-actin and prolactin (PRL) sequences, respectively. Assay of equivalent dilutions of ovarian cDNAs made from 10, 2, and 0.4 micrograms 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.
Delidow
B C
BC
Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington 06032.
Peluso
J J
JJ
White
B A
BA
D000199:Actins / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D004247:DNA / Q000096:biosynthesis; D005260:Female; D005784:Gene Amplification; D010053:Ovary / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains
D000199:Actins; D012333:RNA, Messenger; D004247:DNA