Selection of a stable reference gene for relative copy number profiling of porcine chromosomal genes using SYBR green qPCR

Reference gene with stable copy number is essential for normalization in qPCR based copy number assay. Present study aims to identify a suitable reference gene in pigs for qPCR based relative copy number profiling of chromosomal genes. A total of 30 crossbred pigs of both sexes were cyto-screened and gDNA was extracted from the pigs having numerically normal karyotypes. The copy number stability was studied for 7 genes (FSHB, IL4, IGF1R, TCF24, BRMS1L, ARMC1 and SRSF4) selected on the basis of the chromosomal location, reports of single copy and lack of involvement in structural chromosomal abnormalities. The copy number was estimated from <i>Ct</i> values in 3 technical replicates using 6 animals from either sex for each gene. The stability was evaluated from the variations in Ct values using different (Delta Ct, geNorm, BestKeeper and normFinder) algorithms. While the moderate variation was observed among relative copy number stabilities among the genes, comprehensive ranking revealed the most stable gene for normalization (IGF1R &gt; FSHB &gt; TCF24 &gt; IL4 &gt; ARMC1&gt; SRSF4 &gt; BRMS1L) across the samples. The selected reference gene was validated using DNA of cyto-screened pigs to find out ratio of X and Y chromosome fragments using qPCR based copy number analysis.

在基于定量聚合酶链式反应（qPCR）的拷贝数检测中，拷贝数稳定的参考基因是归一化分析的必要前提。本研究旨在筛选适用于猪的参考基因，以开展染色体基因的qPCR相对拷贝数分析。本研究共纳入30头雌雄各半的杂交猪，对其进行细胞遗传学筛查，并从核型数值正常的个体中提取基因组脱氧核糖核酸（gDNA）。研究选取了7个基因（FSHB、IL4、IGF1R、TCF24、BRMS1L、ARMC1及SRSF4），其筛选依据包括染色体定位、单拷贝相关报道以及未参与染色体结构异常等特征。每个基因选取6头不同性别的个体，通过3次技术重复的循环阈值（Ct）值估算其拷贝数。采用Delta Ct、geNorm、BestKeeper及normFinder四种算法，基于Ct值的变异程度评估各基因的稳定性。尽管各基因的相对拷贝数稳定性存在中等程度的差异，但综合排序结果显示，适用于本实验样本归一化的最稳定基因依次为：IGF1R > FSHB > TCF24 > IL4 > ARMC1 > SRSF4 > BRMS1L。本研究利用经细胞遗传学筛查的猪基因组DNA，通过基于qPCR的拷贝数分析检测X、Y染色体片段的比例，对所选参考基因进行了验证。