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

Abstract

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.

Temporal Transcriptome Dynamics of Longissimus dorsi Reveals the Mechanism of the Differences in Muscle Development and IMF Deposition between Fuqing Goats and Nubian Goats

“…With the increase in the number of physiological groups in research programs, such as breeds, developmental stages, and organizations, many studies have shown that traditional reference genes, such as ACTB and GAPDH , are not suitable for target gene normalization [ 40 , 41 ]. In this study, 16 candidate reference genes ( Table S4 ) were selected from previous reports [ 36 , 38 , 39 ], 9 of which, including GAPDH , were DEGs according to our RNA-Seq data analysis. API5 showed the least variation and was recommended as the best reference gene, following the results obtained for porcine skeletal muscle at 26 different developmental stages by Niu [ 40 ].…”

“…The stable endogenous genes GAPDH [ 36 ], ACTB [ 37 ], HSP90 [ 38 ], PPIB [ 39 ], EIF3K [ 39 ], API5 [ 40 ], DRAP1 [ 41 ], and WSB2 [ 41 ] were selected for data normalization according to previous reports. The stability of the endogenous genes was analyzed using their RNA-Seq data ( Supplementary Table S2 ).…”