Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde‐3‐phosphate dehydrogenase and β‐actin mRNA expression in porcine immune cells and tissues

Abstract

Various "housekeeping" genes are often used as endogenous controls in gene expression experiments. We have cloned from swine, three genes commonly used as endogenous controls in other species and have characterized their relative levels of expression in various porcine tissues and their response to various cell activators. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were readily detected by northern hybridization in various tissues and in alveolar macrophages. The expression of hypoxanthine phosphoribosyltransferase (HPRT) was detected only by northern hybridization of poly-A+ enriched RNA and by reverse transcriptase-polymerase chain reaction (RT-PCR), making it more suitable for highly sensitive detection methods. Expression of GAPDH varied less among tissues than did beta-actin, making it more useful control for comparisons of gene expression between tissues with northern hybridizations. Various treatments of cultured alveolar macrophages differentially affected levels of beta-actin and GAPDH, while HPRT expression was unchanged in alveolar macrophages or spleen cells similarly treated. Therefore, while HPRT can be used as the endogenous control with sensitive detection methods such as RT-PCR, less sensitive detection methods require a more abundant gene such as GAPDH.

Development and evaluation of canine reference genes for accurate quantification of gene expression

“…In view of food safety and animal welfare, in-depth studies on the validation of primers for reference genes in cattle and pigs have been performed [25,26]. The research interest in companion animals such as dogs has been limited mainly to veterinary schools.…”

“…a Because there was not enough data from ACTB, this gene is left out in the liver ranking. G3P and ACTB in cattle and pigs [25,26], especially for highly sensitive analyses such as qPCR. It is possible that the apparent relative unstable expression of ACTB is due to the primers used, and consequently to the amplicon size, rather than a problem of ACTB per se.…”

Homologous and heterologous in vitro regulation of pig pituitary somatostatin receptor subtypes, sst1, sst2 and sst5 mRNA

“…Hence, these parameters must be optimized and tightly controlled. The multiplex RT-PCR developed in this study amplified sst1, 2 and 5 and HPRT (a housekeeping gene (Foss et al 1998)) transcripts from whole pituitary extracts in the same amplification reaction. To determine the cycle numbers that would correspond to the parallel amplification range of all PCR products within each reaction, PCR was performed between 21 and 36 cycles.…”

Semiquantitative reverse transcription-polymerase chain reaction to evaluate the expression patterns of genes involved in the oestrogen pathway

“…Expression levels of two oestrogen independent housekeeping genes were evaluated using cDNA obtained from the Ishikawa cell line: the 2 m gene, which is considered to be highly expressed, and the HPRT gene, which is moderately expressed in mammal cells as compared with other housekeeping genes (Foss et al 1998). The amplification efficiencies of these genes were determined in different PCR rounds (Fig.…”