Housekeeping gene expression in bovine liver is affected by physiological state, feed intake, and dietary treatment

Selection of appropriate housekeeping genes (HKG) for normalization of quantitative PCR data for genes of interest is critical for interpretation of results. Ideally, copy number of the chosen HKG mRNA will not vary with experimental treatments or physiological state in the tissue studied, which improves accuracy in detecting changes in genes of interest. Because of the liver's dynamic role in metabolism, physiological state or dietary treatments could alter mRNA expression of commonly used HKG. Therefore, the objective of this study was to evaluate stability of mRNA expression for a number of candidate HKG in bovine liver across different physiological and dietary experimental conditions during the periparturient period. A publicly available program (geNorm) was used to evaluate expression stability of 8 HKG (beta-actin, glyceraldehyde 3-phosphate dehydrogenase, beta-glucuronidase, peptidylprolyl isomerase A, polyubiquitin, ribosomal protein S9, ribosomal protein L32, and 18S ribosomal RNA) in 91 liver RNA samples. Screened samples included liver from cows in 3 groups: 1) cows receiving a dietary supplement pre- and postpartum (n = 10); 2) cows with clinical or subclinical ketosis (n = 7); and 3) cows consuming different amounts of energy prepartum (n = 74). In group 3, samples from d -65, -30, -14, 1, 14, 28, and 49 relative to parturition were included to enable characterization of HKG mRNA expression across different physiological states. Initial analyses indicated that mRNA for ribosomal protein S9 (RPS9) was one of the most stably expressed across different experiment types. To determine the best gene, 200 bootstrap replications of the original data set were performed to determine if the ranking of RPS9 was superior to the other 7 genes evaluated. Average ranks and estimated standard errors for the top 3 genes were 1.64 +/- 0.06, 3.27 +/- 0.10, and 3.71 +/- 0.12 for RPS9, GAPDH, and beta-actin, respectively. Ribosomal protein S9 was ranked first 59% of the time and was never ranked lower than fifth. The lowest-ranked gene was polyubiquitin, ranked last 46.5% of the time (average rank = 6.85 +/- 0.10). In this study, physiological state, amount of intake, or dietary treatment influenced the mRNA expression of commonly used HKG in bovine liver. Ideally, expression stability should be tested before collection of data in all experiments; however, we have shown that RPS9 mRNA is stable across several physiological and diet-related experimental conditions for dairy cows, making it a good HKG in liver quantitative PCR experiments.