Measurement of Gene Expression by Multiplex Competitive Polymerase Chain Reaction

摘要: Abstract 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) (b) two internal standards consisting single-base mutants GAPDH that cause either loss or gain an Eco RI restriction endonuclease recognition site. RNA extracted human papillomavirus-immortalized bronchial epithelial cell line (BEP2D) reverse transcribed. Serial dilutions DNA were amplified in presence primers quantified amounts mutated standards. The digested with electrophoresed on agarose gel stained ethidium bromide, separating native products. Densitometry performed quantitate bands. Our studies demonstrate this technique measures relative expression precisely reproducibly for done same master mixture dilution Ratios varied less than 25% mean. This will be useful changes expression, particularly when amount study sample is limited level low.