Selection of reference genes for gene expression studies in PBMC from Bama miniature pig under heat stress.

Heat stress decreases immune function and increases disease susceptibility in stressed animals, which are important factors for industry and public health. We investigated the molecular mechanisms underlying heat stress by profiling the expression of target genes involved in the cellular response in the blood of Bama miniature pigs (Sus scrofa domestica) over 21 days with the use of quantitative RT-PCR (qRT-PCR). Reliable standards were established for the normalization of qRT-PCR. Six potential reference genes were ranked by their stability using the geNorm and NormFinder programs. Ribosomal protein L4 (RPL4) and TATA-box-binding protein (TBP) ranked as the two most stably expressed genes, except on day 21 when beta-2-microglobulin (B2M) was the most stable. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal RNA 18S (18SRNA) were discarded as reference genes due to their unstable expression patterns. When B2M and TBP genes were selected as standards in combination, rather than GAPDH, a significant upregulation in Toll-like receptor 2 (TLR2) expression was observed after 21 consecutive days of heat stress. These findings suggest that selection of an ideal reference gene is a key step in comparison of transcription profiles in Bama miniature pigs.