Screening the optimal housekeeping genes (HKGs) of placenta tissues by RNA-sequence and qRT-PCR throughout gestation in goat (Capra Hircus)

Abstract

Selection of stable housekeeping genes (HKGs) is very important for accurate calculation of relative expression levels of target genes by quantitative real-time polymerase chain reaction (qRT-PCR). At present, the appropriate HKGs have not been identified in placental tissues throughout the pregnancy of the goat. In our study, 20 HKGs were tentatively selected from RNA-seq data and previous reports. The cycle threshold (Ct) of HKGs was determined by qRT-PCR in trophoblast membrane and cotyledon villus collected from 38 Dazu Black goats on gestation days of 20, 25, 30, 45, 60, 90, 120, and 150 (birth). The expression stability of the HKGs was analyzed by geNorm, Normfinder, Bestkeeper and Delta Ct algorithms, and comprehensively evaluated by ReFinder and ComprFinder. In addition, the optimal HKGs were further verified by placenta-specific genes (SPP1, VEGFA and PAG6). The 16 candidate HKGs (except POP4, TBP, RNF10, UBC) showed a qualified Ct value, less than 28. Among them, YWHAZ, EIF3K and PPIB showed the most stable expression in placental tissues during early, mid-late pregnancy and postpartum, but the least stable expression was B2M at early and mid-late stage, and PPIB at postpartum. After comprehensive analysis, RPLP0, EIF3K and YWHAZ were found to be the most stable placental HKGs throughout pregnancy. The classical HKGs, ACTB, GAPDH and 18S RNA have unstable expressions and even ranked at the bottom of the list from comprehensive index, suggesting an inappropriate for target gene normalization. Taken together, our study confirmed that YWHAZ, EIF3K, HMBS and RPLP0 may be the optimal HKGs in goat placenta at different stage of pregnancy, which provided a valuable reference of HKGs on functional gene expression detection for further research on placenta development and growth in ruminants.