Selection and validation of suitable reference genes in adipose tissue of Jianzhou Da'er Goat (Capra hircus)

Abstract

Reverse transcription quantitative real-time PCR (RT-qPCR) is a common and high-efficiency technique for detecting the mRNA levels of genes where suitable reference genes were introduced to normalize the data of RT-qPCR. However, little is known about the suitable reference genes in the adipose tissue of Jianzhou da’er goat. Here, six housekeeping genes, including the ACTB, ALAS1, EIF3K, HSP90, RPLP0, and UXT, were selected as candidate reference genes, while the FASN, HSL, LPL, and PID1 were used as the target genes for the validation of the suitability of identified reference genes. After detecting the expression levels of selected genes, the geNorm, NormFinder, and RefFinder softwares were applied to analyze the obtained data of candidate reference genes. The suitability analysis showed that the HSP90 was the most stable candidate reference gene, followed by the ALAS1 and RPLP0 gene. The validation experiment not only verified that the HSP90 was more stable than ACTB as the reference gene, but also confirmed that the HSP90 alone and a combination of HSP90, ALAS1, and RPLP0 could play the same roles in the normalization of target genes. Therefore, we strongly suggested that the HSP90 alone and the combination of three genes (HSP90, ALAS1, and RPLP0) could be used as the suitable reference for normalizing gene expression data in adipose tissue of Jianzhou da’er goat.