Expression stability of putative reference genes in equine endometrial, testicular, and conceptus tissues

Background: Quantitative RT-PCR data are commonly normalized using a reference gene. A reference gene is a transcript which expression does not differ in the tissue of interest independent of the experimental condition. The objective of this study was to evaluate the stability of mRNA expression levels of putative reference genes in three different types of equine tissue, endometrial, testicular, and conceptus tissue. Findings. The expression stability of four (uterine tissue) and six (testicular and conceptus tissue) was assessed using descriptive data analysis and the software programs Normfinder and geNorm. In uterine samples, 18S showed the largest degree of variation in expression while GAPDH, B2M, and ACTB were stably expressed. B2M and GAPDH were identified as the most stably expressed genes in testicular samples, while 18S showed some extent of regulation between samples. Conceptus tissue overall was characterized by very low variability of the transcripts analyzed with GAPDH, YWHZ, and 18S being the most stably expressed genes. Conclusions: In equine endometrium, GAPDH, B2M, and ACTB transcript levels are equally stable, while 18S is less stably expressed. In testes and associated structures, B2M and GAPDH are the transcripts showing the least amount of variation, while in conceptus tissue GAPDH, YWHZ, and 18S were identified as the most suitable reference genes. Overall, transcripts analyzed in conceptus tissue were characterized by less variation than transcripts analyzed in uterine and testicular tissue.