Measurement of interleukin-1 stimulated constitutive prostaglandin G/H synthase (cyclooxygenase) mRNA levels in osteoblastic MC3T3-E1 cells using competitive reverse transcriptase polymerase chain reaction.

To assess regulation of constitutive prostaglandin G/H synthase (PGHS-1) by interleukin-1 (IL-1) in osteoblastic MC3T3-E1 cells, we compared analysis by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) with Northern blot analysis. Using RT-PCR, IL-1 increased PGHS-1 mRNA levels by 1.84 +/- 0.10 or 2.07 +/- 0.17, depending on the method of calculation. Using Northern blot analysis, the effect of IL-1 on PGHS-1 mRNA levels was more variable, and the variability was increased by normalization of PGHS-1 mRNA levels to the housekeeping genes, beta-actin and glyceraldehyde phosphate dehydrogenase (GAPDH), because their mRNA levels were also regulated by IL-1. We conclude that competitive RT-PCR is a reproducible and accurate method for studying small changes in mRNA levels.