Measurement of gene expression by multiplex competitive polymerase chain reaction.
We have developed a polymerase chain reaction (PCR)-based method to measure glutathione peroxidase (GSH-Px) mRNA levels. Expression was measured by multiplex competitive PCR amplification of (a) cDNA from GSH-Px and the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (b) two internal standards consisting of single-base mutants of GSH-Px and GAPDH cDNA that cause either a loss (GSH-Px) or a gain (GAPDH) of an EcoRI restriction endonuclease recognition site. RNA extracted from a human papillomavirus-immortalized human bronchial epithelial cell line (BEP2D) was reverse transcribed. Serial dilutions of cDNA were PCR amplified in the presence of GSH-Px and GAPDH primers and quantified amounts of mutated internal standards. The amplified DNA was restriction digested with EcoRI and electrophoresed on an agarose gel stained with ethidium bromide, separating native from mutated products. Densitometry was performed to quantitate the bands. Our studies demonstrate that this technique measures the relative expression of GSH-Px to GAPDH precisely and reproducibly for studies done with the same master mixture and dilution of internal standards. Ratios of relative gene expression varied less than 25% from the mean. This technique will be useful to measure changes in gene expression, particularly when the amount of study sample is limited or the level of gene expression is low.
Apostolakos
M J
MJ
Department of Medicine, University of Rochester School of Medicine, New York 14642.
Schuermann
W H
WH
Frampton
M W
MW
Utell
M J
MJ
Willey
J C
JC
D001483:Base Sequence; D001980:Bronchi / Q000166:cytology; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D003720:Densitometry; D004996:Ethidium; D005784:Gene Amplification / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D005979:Glutathione Peroxidase / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013698:Templates, Genetic
D018076:DNA, Complementary; D005979:Glutathione Peroxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D004996:Ethidium