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

Yumei Zhou; Xingchun Li; Xinyue Zhang; Minghui Li; Nanjian Luo; Yongju Zhao

doi:10.3390/ani13121897

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

Animals (Basel). 2023 Jun 6;13(12):1897. doi: 10.3390/ani13121897.

Authors

Yumei Zhou^{1

2}, Xingchun Li^{1

2}, Xinyue Zhang^{1

2}, Minghui Li^{1

2}, Nanjian Luo^{1

2

3}, Yongju Zhao^{1

2}

Affiliations

¹ College of Animal Science and Technology, Southwest University, Chongqing 400715, China.
² Chongqing Key Laboratory of Herbivore Science, Chongqing Key Laboratory of Forage and Herbivore, Chongqing Engineering Research Center for Herbivores Resource Protection and Utilization, Chongqing Herbivore Engineering Research Center, Chongqing 400715, China.
³ School of Preclinical Medicine, Zunyi Medical University, Zunyi 563000, China.

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.

Keywords: RNA-sequence; goat; the candidate housekeeping genes; uterus caruncle.

Grants and funding

This work was supported by the National Natural Science Foundation of China (No. 31772564), Chongqing’s Modern Agricultural Industry Technology System Program for Herbivore (2023[14]), Collection, Utilization and Innovation of Animal Resources by Research Institutes and Enterprises of Chongqing (No. qnyncw-kqlhtxm) and Research and Innovation Project of Chongqing Graduate Students (No. CYB17073).