Department of Gastroenterology, The First People's Hospital of Jiande, Hangzhou, 311600, China.
Department of Biological Repositories, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
Sci Rep. 2023 Oct 18;13(1):17777. doi: 10.1038/s41598-023-45174-4.
Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common method to determine mRNA expression, and Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that a panel of reference genes for RT-qPCR is conducive to obtaining accurate results. This study aimed to screen and verify the optimal panel of reference genes in colorectal cancer (CRC) and normal colonic cell lines. In the study, eight candidate reference genes (GAPDH, ACTB, 18S, PPIA, B2M, SDHA, GUSB, and YWHAZ) were selected for RT-qPCR to detect their expression in NCM460, HT29, HCT116, SW480, SW620, DLD-1, LOVO and RKO cell lines. The stability of reference genes and the optimal panel were evaluated by geNorm, NormFinder, and BestKeeper software. As results, the expression levels of candidate reference genes differed in the colonic epithelial cell lines, and the number of optimal panel of reference genes is two. B2M and YWHAZ were the two most stable reference genes for NCM460, HCT116, SW620, LOVO, and RKO cell lines, while only one of B2M and YWHAZ was most stable in HT29 and SW480 cells. In DLD-1 cells, the stability of B2M and YWHAZ ranked 3rd and 6th, PPIA and GUSB were the most stable two. Furthermore, the YWHZA + B2M performed smaller intragroup differences than other panel or single reference gene. In conclusion, this study indicates the optimal panel of reference genes is YWHZA + B2M for the NCM460, HCT116, SW620, LOVO, RKO, SW480, and HT29 cell lines, but it is PPIA + GUSB in DLD-1 cell lines.
实时定量聚合酶链反应（RT-qPCR）是确定 mRNA 表达最常用的方法，最小信息发布实时定量 PCR 实验（MIQE）提出 RT-qPCR 参考基因的面板有利于获得准确的结果。本研究旨在筛选和验证结直肠癌（CRC）和正常结肠细胞系中最佳的参考基因组合。在研究中，选择 8 个候选参考基因（GAPDH、ACTB、18S、PPIA、B2M、SDHA、GUSB 和 YWHAZ）进行 RT-qPCR 检测，以检测它们在 NCM460、HT29、HCT116、SW480、SW620、DLD-1、LOVO 和 RKO 细胞系中的表达。通过 geNorm、NormFinder 和 BestKeeper 软件评估参考基因的稳定性和最佳面板。结果，候选参考基因在结肠上皮细胞系中的表达水平不同，最佳参考基因面板的数量为两个。B2M 和 YWHAZ 是 NCM460、HCT116、SW620、LOVO 和 RKO 细胞系中最稳定的两个参考基因，而 HT29 和 SW480 细胞中只有一个 B2M 和 YWHAZ 最稳定。在 DLD-1 细胞中，B2M 和 YWHAZ 的稳定性分别排在第 3 位和第 6 位，PPIA 和 GUSB 是最稳定的两个。此外，与其他面板或单个参考基因相比，YWHAZ+B2M 具有更小的组内差异。总之，本研究表明，对于 NCM460、HCT116、SW620、LOVO、RKO、SW480 和 HT29 细胞系，最佳参考基因组合是 YWHZA+B2M，但在 DLD-1 细胞系中是 PPIA+GUSB。
