Screening of Candidate Housekeeping Genes in Uterus Caruncle by RNA-Sequence and qPCR Analyses in Different Stages of Goat (Capra hircus)

Simple Summary The uterus is an important organ in the reproductive system of most female mammals. The normal growth and development of ruminant uterus caruncles are crucial to maintain gestation and fetal health in goats. The aim of this study was to screen a set of candidate housekeeping genes (HKGs) in Carpra hircus uterus caruncles. Four programs and one comprehensive index were applied to assess the stability of 22 selected HKGs expression during non-pregnant and pregnancy stages. The most and least stable HKGs were used to normalize the target genes expression of SPP1, VEGFA, and PAG8. Traditional reference genes were not suitable for target gene normalization, while PPIB and EIF3K, POP4, PPIA, and SDHA showed the least variation and were recommended as the best HKGs during the nonpregnant stage and the whole stages of goat uterus caruncle tissue, respectively. This study found the suitable HKGs in uterus caruncle tissues at different stages of non-pregnancy and pregnancy. The uterus is a critical pregnancy organ for mammals. The normal growth and development of ruminant uterus caruncles are crucial to maintain gestation and fetal health in goats. Quantitative real-time polymerase chain reaction (qRT-PCR) is a reliable tool to study gene expression profiling for exploring the intrinsic mechanism underlying the conversion process of uterus caruncle tissue. However, the candidate housekeeping genes (HKGs) are required for normalizing the expression of function genes. In our study, 22 HKGs were selected from analyzing transcriptome data at non-pregnancy and pregnancy processes and previous reports about HKGs in goat tissues. We assessed them for expression suitability in 24 samples from uterus tissues at 15 non-pregnant days (Stage 1), early (Stage 2), and medium-later pregnant days (Stage 3). The expression stability of these genes was evaluated by using geNorm, Normfinder, Bestkeeper, and Delta Ct algorithms and, comprehensively, by ReFinder. In addition, the most and least stable HKGs were used to normalize the target genes expression of SPP1, VEGFA, and PAG8. It was found that traditional reference genes, such as ACTB and GAPDH, were not suitable for target gene normalization. In contrast, PPIB selected from RNA sequencing data and EIF3K selected from previous references showed the least variation and were recommended as the best HKGs during the nonpregnant stage and the whole stages of goat uterus caruncle tissue, respectively. It is the first time the HKGs genes in uterus during the non-pregnant day and throughout the total pregnancy have been explored. These findings found suitable HKGs in uterus caruncle tissues at various stages of non-pregnancy and pregnancy; these can be useful for gene expression studies to reveal the molecular mechanisms of uterus development in goats.

Automated summary

This study aimed to screen candidate housekeeping genes in Carpra hircus uterus caruncles. The stability of 22 selected genes was assessed during non-pregnant and pregnancy stages. PPIB and EIF3K were found to be the most stable reference genes and recommended for use in nonpregnant and whole stages of goat uterus caruncle tissue. This study identified suitable reference genes for gene expression studies in uterus caruncle tissues at various stages of non-pregnancy and pregnancy.