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

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, <i>ALAS1</i>, <i>EIF3K</i>, <i>HSP90</i>, <i>RPLP0</i>, and UXT, were selected as candidate reference genes, while the FASN, <i>HSL</i>, <i>LPL</i>, and <i>PID1</i> 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, <i>ALAS1</i>, 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, <i>ALAS1</i>, and RPLP0) could be used as the suitable reference for normalizing gene expression data in adipose tissue of Jianzhou da’er goat.逆转录实时荧光定量聚合酶链反应（RT-qPCR）是检测基因mRNA水平的常用高效技术，该技术需引入合适的内参基因以对RT-qPCR数据进行归一化处理。然而，目前关于简州大耳羊脂肪组织中适宜内参基因的研究尚少。本研究选取6种持家基因作为候选内参基因，包括ACTB、ALAS1、EIF3K、HSP90、RPLP0及UXT；同时选取FASN、HSL、LPL、PID1作为靶基因，用于验证所筛选内参基因的适用性。在检测目标基因的表达水平后，本研究采用geNorm、NormFinder及RefFinder三款软件对候选内参基因的表达数据进行分析。适用性分析结果显示，HSP90是稳定性最优的候选内参基因，其次为ALAS1与RPLP0。验证实验不仅证实HSP90作为内参基因的稳定性优于ACTB，同时确认单独使用HSP90，或联合使用HSP90、ALAS1与RPLP0，均可在靶基因的归一化处理中发挥同等作用。综上，本研究推荐单独使用HSP90，或联合使用HSP90、ALAS1与RPLP0，作为简州大耳羊脂肪组织中基因表达数据归一化处理的合适内参基因。