ORIGINAL ARTICLE: Putative reference genes for gene expression studies in propionate and β-hydroxybutyrate treated bovine adipose tissue explants

Abstract

Accurate gene expression normalization using a stable reference gene (RG) improves the reliability of quantitative real-time PCR (qPCR) results. Therefore, a validation of RGs should be done before their use. Only few studies on RGs have been done in cattle, and none in bovine adipose tissue (AT) explants, therefore, we characterize the effects of an in vitro treatment with propionate and β-hydroxybutyric acid (BHB) on the mRNA content of these RGs comparing the output data from the geNorm™ and the Normfinder(©) program. geNorm™ and Normfinder(©) estimated the most stable RGs in the following sequence for subcutaneous and for retroperitoneal AT explants treated with propionate: low density lipoprotein receptor-related protein 10 (LRP10) > hippocalcin-like 1 (HPCAL1) > glyceraldehyde-phosphate-dehydrogenase (GAPDH) > ribosomal protein S9 (RPS9) > RNA polymerase II (Pol II) > beta2 actin (ACTB) > 18S ribosomal RNA (18S rRNA). BHB treated AT explants yielded a different stability ranking for RGs using geNorm™: HPCAL1, GAPDH > Pol II > LRP10 > ACTB > RPS9 > 18S rRNA. Normfinder(©) estimated a different stability ranking for the RGs as shown in the following sequence for subcutaneous and retroperitoneal AT explants treated with BHB: HPCAL1 > Pol II > GAPDH > ACTB > LRP10 > RPS9 > 18S rRNA. Subsequent pairwise analysis of variation of RGs using geNorm™ suggested that LRP10, HPCAL1 and GAPDH should be used for accurate normalization of subcutaneous and retroperitoneal AT explants treated with propionate, while HPCAL1, GAPDH and Pol II should be used for BHB treatment.