Transcriptome-based selection and validation of optimal house-keeping genes for skin research in goats (Capra hircus)

In quantitative real-time polymerase chain reaction (qRT-PCR) experiments, accurate and reliable target gene expression results are dependent on optimal amplification of house-keeping genes (HKGs). RNA-seq technology offers a novel approach to detect new HKGs with improved stability. Goat (Capra hircus) is an economically important livestock species and plays an indispensable role in the world animal fiber and meat industry. Unfortunately, uniform and reliable HKGs for skin research have not been identified in goat. Therefore, this study seeks to identify a set of stable HKGs for the skin tissue of C. hircus using high-throughput sequencing technology.

Based on the transcriptome dataset of 39 goat skin tissue samples, 8 genes (SRP68, NCBP3, RRAGA, EIF4H, CTBP2, PTPRA, CNBP, and EEF2) with relatively stable expression levels were identified and selected as new candidate HKGs. Commonly used HKGs including SDHA and YWHAZ from a previous study, and 2 conventional genes (ACTB and GAPDH) were also examined. Four different experimental variables: (1) different development stages, (2) hair follicle cycle stages, (3) breeds, and (4) sampling sites were used for determination and validation. Four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method) and a comprehensive algorithm (ComprFinder, developed in-house) were used to assess the stability of each HKG. It was shown that NCBP3 + SDHA + PTPRA were more stably expressed than previously used genes in all conditions analysis, and that this combination was effective at normalizing target gene expression. Moreover, a new algorithm for comprehensive analysis, ComprFinder, was developed and released.

This study presents the first list of candidate HKGs for C. hircus skin tissues based on an RNA-seq dataset. We propose that the NCBP3 + SDHA + PTPRA combination could be regarded as a triplet set of HKGs in skin molecular biology experiments in C. hircus and other closely related species. In addition, we also encourage researchers who perform candidate HKG evaluations and who require comprehensive analysis to adopt our new algorithm, ComprFinder.