Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR

The effects of serum on the expression of four commonly used housekeeping genes were examined in serum-stimulated fibroblasts in order to validate the internal control genes for a quantitative RT-PCR assay. NIH 3T3 fibroblasts transfected with an inducible chimeric gene were serum-starved for 24 h and then induced with 15% serum for 8 h. Serum did not alter the amount of total RNA that was expressed in the cells, however, the amount of mRNA significantly increased over time with serum-stimulation. Both messenger and total RNA from each of the time points were reverse transcribed under two different conditions; one in which the reactions were normalized to contain equal amounts of RNA and another series of reactions that were not normalized to RNA content. The resulting cDNA was amplified by real-time, quantitative PCR using gene-specific primers for β-actin, β-2 microglobulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA. The expression of β-actin and GAPDH increased up to nine- and three-fold, respectively, under all conditions of reverse transcription ( P