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

Abstract

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<0.01). The expression of 18S rRNA increased with serum-stimulation when the cDNA synthesized from non-normalized, total RNA was assayed (P<0.01) but not when the reverse transcriptions were normalized to RNA content (P>0.05). The expression of β-2 microglobulin increased up to two-fold when assayed from cDNA synthesized from non-normalized mRNA, but was unaffected by serum when the reverse transcriptions were normalized to mRNA. β-2 Microglobulin expression was found to be directly proportional to the amount of mRNA that was present in non-normalized reverse transcription reactions. Thus, β-2 microglobulin and 18S rRNA are suitable internal control genes in quantitative serum-stimulation studies, while β-actin and GAPDH are not. The internal control gene needs to be properly validated when designing quantitative gene expression studies.

Introduction

The response of mammalian cells to serum is a useful model to study complex cellular processes that are influenced by extracellular signaling molecules (reviewed in [1]). Cultured murine fibroblasts (e.g. NIH 3T3, Swiss 3T3, Balb/c 3T3) are particularly useful for these studies. Culture media supplemented with 10–20% serum provides the fibroblasts with the necessary polypeptide growth factors. Serum-starvation, or reducing the amount of serum in the culture media, forces the fibroblasts to enter a quiescent or G₀ phase of the cell cycle. Quiescent cells may be stimulated to reenter the cell cycle by adding 10–20% serum. Serum-stimulation of quiescent fibroblasts induced numerous genes including transcription factors (c-Fos, c-Myc), cytoskeletal and extracellular matrix proteins (β-actin, fibronectin), enzymes (MAP kinase phosphatase-1, nitric oxide synthase) and many others [1]. Qualitative analysis of over 8600 different genes using cDNA microarray technology revealed that genes could be clustered into several different groups based upon their response to serum [2].

Quantitative gene expression assays are typically referenced to an internal control gene such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin, to account for differences in RNA load. The amount of RNA assayed may fluctuate due to differences in tissue mass, cell number, experimental treatment or RNA extraction efficiency. Ideally, the conditions of the experiment should not influence the expression of the internal control gene. Selecting an internal control gene for quantitative gene expression studies of serum-stimulated fibroblasts is difficult because of the ubiquitous effect that serum has on gene expression [1], [2]. We initiated mRNA stability studies using NIH 3T3 fibroblasts that were stably transfected with the serum-inducible fos–glo–myc chimeric gene [3]. Fibroblasts transfected with these chimeric genes are routinely used to study mRNA decay. In order to validate a quantitative RT-PCR assay to study decay of the fos–glo–myc mRNA, we examined the effect of serum on the expression of several candidate internal control genes.

We report a detailed quantitative analysis of the expression of four commonly used housekeeping genes during the early response to serum and the appropriate internal controls to use in quantitative serum-stimulation studies. The methodology reported here may be applied to other quantitative gene-expression studies to evaluate the effects of experimental treatment on the expression of potential internal control genes.