Identification of internal reference genes for porcine immature Sertoli cells under heat stress.

Identification of stably expressed gene(s) as internal reference(s) for different experimental conditions is key to the accurate normalization and quantification of target transcripts. Previously, our RNA-seq study showed that Hprt1, Actb, and 18S rRNA abundances were all significantly altered in porcine immature Sertoli cells (iSCs) during acute heat stress (HS). In the current study, we aimed to identify stable reference gene(s) to study the gene expression dynamics of quick and delayed responses after acute HS treatment of porcine iSCs. A total of six genes previously used in pig testis or Sertoli cells (Hprt1, Top2b, Actb, Rpl32, Gapdh, and 18S rRNA) were chosen to perform RT-qPCR for the control (before acute HS), HS0.5 (acute HS at 43°C for 0.5 h), and HS0.5-R36 (36 h recovery following acute HS) groups. The stability of candidate reference genes was examined by the GeNorm, NormFinder, BestKeeper and Comparative ΔCt methods, and RefFinder to obtain the final rank. Rpl32 and Actb were the two most stable internal reference genes as found by all methods, whereas Hprt1 and 18S rRNA were the two most unstable as ranked by RefFinder. Moreover, expression of six target mRNAs (Ccn1, Ccnb1, Eif4g1, Hdac6, Plk2, and Ptma) was normalized using Rpl32, Actb, or the combination of Rpl32 and Actb, respectively. Therefore, our findings that the most suitable internal references are Rpl32 and Actb provide useful information for further functional investigation on genes regulating the acute HS of porcine iSCs.