Abstract #W82

# W82
Identification of novel real-time quantitative PCR reference genes for bovine corpus luteum via whole-transcriptome RNA sequencing.
M. A. Mezera*1, L. Wenli2, D. J. Koch2, A. Edwards2, C. A. Gammara1, R. S. Gennari1, V. E. Gomez-Leon1, R. Reis Domingues1, A. D. Beard1, M. C. Wiltbank1, 1University of Wisconsin- Madison, Madison, WI, 2USDA Dairy Forage Research Center, Madison, WI.

Real-time quantitative PCR (RTqPCR) is a valuable tool to study gene expression in tissues. However, tissues from dynamic physiological states pose challenges to normalization, and thus analysis, as traditional reference genes may be unstable across physiologic states. The corpus luteum (CL) is dynamic throughout luteolysis and maintenance in pregnancy. Thus, there is a need to identify stable genes throughout physiologic conditions for use as references genes in RTqPCR analysis. Stable genes were discovered with whole transcriptome RNA sequencing (RNaseq) and validated with RT-qPCR. CL biopsies collected from 5 states were subjected to RNA-seq analysis: CL from pregnant animals during (n = 5, d 20 ± 0) and after (n = 4, d 55.3 ± 3.4) secretion of interferon-tau, and CL before (n = 10), during (n = 8), and after (n = 5) functional luteolysis (luteolytic progression based on circulating progesterone and prostaglandin F2 α metabolite). Potential reference genes were identified by ANOVA analysis of normalized read counts calculated by Cufflinks. Seventy-seven genes had a p-value greater than 0.1 and standard deviation less than 20% of mean read count. Genes were further analyzed with the R/Bioconductor package DEseq2 by randomly assigning samples to 2 groups. The 6 genes with the highest p-values were further analyzed (RPL4, UQCRFS1, COX4I1, RPS4X, SSR3, and CST3) with RT-qPCR analysis of an independent set of CL samples from first (n = 4) and second month (n = 4) pregnant cows, and CL before (n = 5), during (n = 5), and after (n = 5) functional luteolysis. Gene stability from PCR was calculated with the algorithms geNorm and NormFinder. Four genes were consistently more stable than ACTB and GAPDH, the most common reference genes in bovine CL literature, regardless of physiologic state based on RT-qPCR analysis: RPL4, COX4I1, SSR3, and RPS4X. In CL tissue from pregnancy, CST3 had highest stability. The identification of these novel reference genes will aid accurate normalization of RT-qPCR results. Furthermore, analyses shed light into the effects of luteolysis and pregnancy on stability of gene expression in the CL.

Key Words: corpus luteum, PCR