A polymerase chain reaction assay for simultaneous detection and quantitation of proto-oncogene and GAPD mRNAs in different cell growth rates

• Published in: Cell proliferation Vol. 30; no. 6; pp. 271 - 282
• Main Authors: Goswami, P. C., Albee, L. D., Spitz, D. R., Ridnour, L. A.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.06.1997; John Wiley & Sons, Inc
• Subjects: Aldehydes; Animals; Assaying; Cell Division; Cell Line; Cricetinae; Cricetulus; Embryos; Exposure; Fibroblasts; Glyceraldehyde-3-Phosphate Dehydrogenases - genetics; Glyceraldehyde-3-Phosphate Dehydrogenases - isolation & purification; Growth conditions; Growth rate; Mice; Mice, Inbred C3H; Myc protein; Nitric oxide; Nitric Oxide - pharmacology; Northern blotting; Oligonucleotides; Oncogenes; Original Paper; Polymerase chain reaction; Polymerase Chain Reaction - methods; Proto-Oncogene Proteins c-fos - genetics; Proto-Oncogene Proteins c-fos - isolation & purification; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - isolation & purification; Quantitation; Reference Standards; Ribonucleic acid; RNA; RNA, Messenger - analysis; RNA-directed DNA polymerase
• ISSN: 0960-7722; 1365-2184
• DOI: 10.1046/j.1365-2184.1997.00093.x

A reverse transcriptase followed by a polymerase chain‐reaction (RT‐PCR) assay was developed for the simultaneous detection and quantitation of proto‐oncogene (c‐fos and c‐myc) mRNAs using an internal standard mRNA glyceraldehyde‐6‐phosphate dehydrogenase (GAPD). Total cellular RNA was reverse transcribed and PCR amplified with oligonucleotide primers specific to GAPD and either c‐fos or c‐myc genes. In contrast to Northern blot analysis, the RT‐PCR assay is rapid and sensitive enough to quantitate specific proto‐oncogene levels from as little as 12–25 ng of total cellular RNA. The reliability of the assay was tested by measuring c‐fos and c‐myc expression in C3H 10T1/2 mouse embryo fibroblast cells under two different growth states: (a) quiescent cell entry into the proliferative cycle, and (b) plateau phase. Furthermore, the assay was used in measuring variations in c‐fos or c‐myc expression in HA‐1 hamster cells following exposure to the cellular stressing agent, nitric oxide. In serum‐stimulated cells, the RT‐PCR measurements of transient increase in c‐fos (16‐fold at 30 min) and c‐myc (10‐fold at 1 h) mRNA levels were comparable to previously reported results in the literature using a Northern blotting assay. In addition, a two‐ to fivefold increase in c‐fos mRNA levels was observed in plateau phase cells when compared to log phase growth. Furthermore, a transient increase in c‐fos mRNA levels (threefold at 2 h) was also observed following cells' exposure to the stressing agent nitric oxide. These results suggest that the multiplex RT‐PCR assay represents a significant improvement over current methods to quantitate specific cellular mRNAs under different growth conditions or following environmental insults.