Selection and validation of suitable reference genes in skin tissue of Liaoning cashmere goat during hair follicle cycle

In quantitative real-time PCR assay, the appropriate reference genes are essential to reduce the potential errors in quantification of gene expression. To date, there is not a well-defined and validated set of reference genes for skin tissue of cashmere goat. In the present work, based on the geNorm and NormFinder procedures, the expression stabilities of eight candidate housekeeping genes were assessed in skin tissue of Liaoning cashmere goat during hair follicle cycle including 18S, ACTB, B2M, GAPDH, TBP, UBC, YWHAZ, and SDHA. Both geNorm and NormFinder identified SDHA, YWHAZ and UBC as the most stable genes in skin of Liaoning cashmere goat. Also, we further validated the suitability of SDHA, YWHAZ and UBC in combination as references in skin of Liaoning cashmere goat via detecting the relative expression of TGFβR2, BMP2, MSX2 and Hoxc13 as target genes. Significant differences were revealed in the relative expression of TGFβR2, BMP2, MSX2, and Hoxc13 at anagen and telogen when a combination of SDHA, YWHAZ and UBC, versus single SDHA were used as reference genes. These results suggested that the combined use of three genes (SDHA, YWHAZ, and UBC) as references would be more reliable than that of a single SDHA for Q-PCR data normalization in skin tissue of Liaoning Cashmere goat. Therefore, we strongly recommended that these three genes can be used as a combined reference for normalization of gene expression in future longitudinal studies on skin of Liaoning cashmere goat associated with import cashmere traits.