Evaluation of various housekeeping genes for their applicability for normalization of mRNA expression in...

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is an extremely sensitive, convenient and rapid method to measure mRNA levels in cells and tissues, and is gaining popularity in toxicology. To correct for sample-to-sample variation, normalization of the expression data is required. The conventional way to perform normalization is to select a reference gene whose expression is believed to remain stable across all experimental conditions, then relate the concentrations of gene(s) of interest to those of this housekeeping gene. Since recent evidence shows that some housekeeping genes are actually not as refractory to experimental manipulations as previously thought, we validated a large number (18) of commonly used housekeeping genes for acute toxicity studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an extremely potent environmental toxin known to regulate a wide variety of genes. Microarray and qRT-PCR analyses coherently demonstrated that about 50% of the housekeeping genes examined were responsive to TCDD in rat liver with the magnitudes of change up to nearly 10-fold. Extension of the study to spleen and hypothalamus verified that phosphoglycerate kinase 1 (Pgk1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) retained their basal expression levels in all experimental settings, although body weight loss-generated repression may mask a slight induction of GAPDH by TCDD in liver. These findings show that normalization genes for qRT-PCR must be carefully validated in advance, especially if the study involves a potent modifier of gene expression.