Selection of reference genes for studies of porcine endometrial gene expression on gestational day 12

Comparing gene expression patterns in the endometrium on gestational day 12 (GD12) between Erhualian (ER) and Landrace × Large White (LL) pigs is helpful to understand the biological mechanisms of fecundity. Selecting genes that have stable expression levels as the internal standards in a comparative study is essential for identifying real gene-specific variation by quantitative RT-PCR (qRT-PCR). Five genes expressed in sow endometria on GD12 were evaluated for their suitability as internal control for relative quantification by qRT-PCR. These genes were beta-actin (ACTB), beta-2-microglobulin (B2M), phosphoglycerate kinase 1 (PGK1), RNA polymerase II polypeptide G (RPG), and ribosomal protein S20 (RPS20), which represent different functional classes. Our results indicated that ACTB, B2M, and PGK1 were not suitable as internal standards for normalization because of their huge variability between the two breeds. RPS20 and RPG were most stable, and the former is recommended to serve as the internal standard when the use of multiple housekeeping genes is unpractical.

► Reference gene selection in endometrial gene expression study was investigated. ► Housekeeping genes are not always suitable for internal control in qRT-PCR. ► RPS20 and RPG were most stably expressed in endometria of the two breeds. ► RPS20 is recommended when the use of multiple reference genes is unpractical. ► Equal expression is implicitly proposed as a criterion for reference gene selection.