Investigation of gene stability in equine luteal tissue during mid-diestrus phase and early pregnancy - Research Article.

Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a technique that allows for the semi-quantification of mRNA transcripts present within a tissue of interest. Differences in the relative abundance of mRNA between samples detected by RT-qPCR require normalization with a reference gene or genes whose transcript abundance is stable within the tissue of interest independent of experimental conditions. In the field of equine reproductive studies, ACTB, GAPDH and B2M genes are the most widely used as reference genes for the normalization of RT-qPCR results. However, recent studies have demonstrated that these genes may have drastically varied expression levels in different tissues and various physiological states. Therefore, the aim of this study was to examine different putative reference genes in equine corpus luteum samples in pregnant and non-pregnant, mid-diestrus, animals. The stability of genetic expression was evaluated via three stability software analyses (GeNorm, NormFinder and BestKeeper). We hypothesized that the commonly used reference genes (ACTB, GAPDH and B2M) would be the most stably expressed genes in equine corpus luteum samples in both pregnant and non-pregnant mares. COX4I1 and SRP14 were both found to be among the top three most stable genes of all samples for all methods, though the ranking of stability changed depending on the software used. When assessing the least stably expressed genes, the commonly used reference genes were frequently identified across the three software. Commonly used reference genes (ACTB, GAPDH, B2M) for RT-qPCR normalization were amongst the least stably expressed genes in equine corpus luteum samples of pregnant and nonpregnant mares at days 11 and 13 of gestation. The most stably expressed putative reference genes using 3 different analysis modalities were SRP14, COX4I1, RPL13 and RPL4. Exploration of putative reference genes should be considered when investigating dynamic endocrine organs such as those used in reproductive studies.