Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR
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.
Section snippets
Chemicals
Tissue culture reagents, random hexamers and MMLV reverse transcriptase were from Life Technologies (Gaithersberg, MD, USA). Hygromycin B was purchased from Sigma (St. Louis, MO, USA). The RNeasy Mini RNA isolation kit was from Qiagen (Valencia, CA, USA). The SYBR green I PCR kit was purchased from PE Biosystems (Foster City, CA, USA). SYBR green II was purchased from Molecular Probes (Eugene, OR, USA).
Tissue culture
NIH 3T3 fibroblasts stably transfected with the fos–glo–myc chimeric gene were generously
Relationship between serum stimulation and cellular RNA levels
NIH 3T3 fibroblasts were forced to enter a quiescent state by a 24-h period of serum-starvation in 0.5% serum. The cells were stimulated with 15% serum and samples were collected over 8 h. Serum-stimulation produced a steady increase in mRNA synthesis, increasing to a maximum of two-fold over 8 h (Fig. 1). The relationship between the time of serum-stimulation and mRNA content was statistically significant (P<0.001, Table 2). No significant relationship was observed between the
Appropriate internal control genes for quantitative serum-stimulation studies
Ideally, the internal control gene for quantitative gene expression studies should not be influenced by the conditions of the experiment. We demonstrate that serum-stimulation influenced the expression of several commonly used housekeeping genes. Serum stimulation for 8 h significantly increased the expression of β-actin and GAPDH (Fig. 2, Fig. 3 and Table 3). Furthermore, serum stimulated the expression of β-actin and GAPDH to a greater extent than it influenced cellular mRNA levels (Fig. 4).
Acknowledgements
We thank Dr. Jeff Ross for providing us with the 3T3 fos–glo–myc cells and Dr. David A. Sclar for his assistance with the statistical analysis.
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