Identification of stable genes in the corpus luteum of lactating Holstein cows in pregnancy and luteolysis: Implications for selection of reverse-transcription quantitative PCR reference genes

摘要

dynamic endocrine tissue vital for pregnancy maintenance,fertility, and cyclicity. Understanding processesunderlying luteal physiology is therefore necessary toincrease reproductive efficiency in cattle. A commontechnique for investigating luteal physiology is reversetranscriptionquantitative PCR (RT-qPCR), a valuabletool for quantifying gene expression. However,reference-gene-based RT-qPCR quantification methodsrequire utilization of stably expressed genes to accuratelyassess mRNA expression. Historically, selectionof reference genes in cattle has relied on subjectiveselection of a small pool of reference genes, many ofwhich may have significant expression variation amongdifferent tissues or physiologic states. This is particularlyconcerning in dynamic tissues such as the CL,with its capacity for rapid physiologic changes duringluteolysis, and likely in the less characterized periodof CL maintenance during pregnancy. Thus, there isa clear need to identify reference genes well suitedfor the bovine CL over a wide range of physiologicalstates. Whole-transcriptome RNA sequencing standsas an effective method to identify new reference genesby enabling the assessment of the expression profile ofthe entire pool of mRNA transcripts. We report theidentification of 13 novel putative reference genes usingRNA sequencing in the bovine CL throughout earlypregnancy and luteolysis: RPL4, UQCRFS1, COX4I1,RPS4X, SSR3, CST3, ZNF266, CDC42, CD63, HIF1A,YWHAE, EIF3E, and PPIB. Independent RT-qPCRanalyses were conducted confirming expression stabilityin another set of CL tissues from pregnancy andregression, with analyses performed for 3 groups ofsamples: (1) all samples, (2) samples from pregnancyalone, and (3) samples throughout the process of CLregression. Seven genes were found to be more stablein all states than 2 traditional reference genes (ACTBand GAPDH): RPS4X, COX4I1, PPIB, SSR3, RPL4,YWHAE, and CDC42. When CL tissues from pregnantanimals alone were analyzed, CST3, HIF1A, and CD63were also identified as more stable than ACTB andGAPDH. Identification of these new reference geneswill aid in accurate normalization of RT-qPCR results,contributing to proper interpretation of gene expressionrelevant to luteal physiology. Furthermore, our analysissheds light on the effects of luteolysis and pregnancy onthe stability of gene expression in the bovine CL.