Cellular Biology, Research and Development Department, Ezequiel Dias Foundation, Belo Horizonte, Minas Gerais 30510-010, Brazil; Translacional Research Laboratory, Mário Penna Institute, Belo Horizonte, Minas Gerais 31270-901, Brazil.
Cellular Biology, Research and Development Department, Ezequiel Dias Foundation, Belo Horizonte, Minas Gerais 30510-010, Brazil; Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil.
Acta Histochem. 2022 Jan;124(1):151821. doi: 10.1016/j.acthis.2021.151821. Epub 2021 Nov 30.
The identification of the best reference gene is a critical step to evaluate the relative change in mRNA expression of a target gene by RT-qPCR. In this work, we evaluated nineteen genes of different functional classes using Real Time Human Reference Gene Panel (Roche Applied Sciences), to identify the internal housekeeping genes (HKGs) most suitable for gene expression normalization data in human cell lines. Normal cell lines CCD-19LU (lung fibroblast), HEK-293 (epithelial cell of embryonic kidney), WI-26 VA4 (lung fibroblast), and human cancer cells, BT-549 (breast cancer), Hs 578T (breast cancer), MACL-1 (breast cancer), HeLa (cervical carcinoma), U-87 MG (glioblastoma/astrocytoma), RKO-AS45-1 (colorectal carcinoma), and TOV-21G (ovarian adenocarcinoma) were cultivated according to manufacturer's protocol. Twelve candidate reference genes were commonly expressed in five cell lines (CCD-19Lu, HEK-293, RKO-AS45-1, TOV-21G, and U-87 MG). To verify the expression stability, we used the RefFinder web tool, which integrates data from the computational programs Normfinder, BestKeeper, geNorm, and the comparative Delta-Ct method. The ACTB was the most stable reference gene to the CCD-19Lu and HEK-293 cells. The best combination of HKGs for the RKO-AS45-1 and TOV-21G cell lines were B2M/GAPDH and PBGD/B2M, respectively. For the U-87 MG cells, GAPDH and IPO8 were the most suitable HKGs. Thus, our findings showed that it is crucial to use the right HKGs to precise normalize gene expression levels in cancer studies, once a suitable HKG for one cell type cannot be to the other.
通过 RT-qPCR 评估靶基因 mRNA 表达的相对变化时，鉴定最佳参考基因是关键步骤。在这项工作中，我们使用实时人类参考基因面板（罗氏应用科学公司）评估了 19 个具有不同功能类别的基因，以鉴定最适合人类细胞系基因表达正常化数据的内部管家基因（HKGs）。正常细胞系 CCD-19LU（肺成纤维细胞）、HEK-293（胚胎肾上皮细胞）、WI-26 VA4（肺成纤维细胞）和人类癌细胞 BT-549（乳腺癌）、Hs 578T（乳腺癌）、MACL-1（乳腺癌）、HeLa（宫颈癌）、U-87 MG（胶质母细胞瘤/星形细胞瘤）和 RKO-AS45-1（结直肠癌）以及 TOV-21G（卵巢腺癌）按照制造商的方案进行培养。12 个候选参考基因在 5 个细胞系（CCD-19Lu、HEK-293、RKO-AS45-1、TOV-21G 和 U-87 MG）中共同表达。为了验证表达稳定性，我们使用 RefFinder 网络工具，该工具整合了来自计算程序 Normfinder、BestKeeper、geNorm 和比较 Delta-Ct 方法的数据。ACTB 是 CCD-19Lu 和 HEK-293 细胞中最稳定的参考基因。对于 RKO-AS45-1 和 TOV-21G 细胞系，最佳 HKG 组合分别为 B2M/GAPDH 和 PBGD/B2M。对于 U-87 MG 细胞，GAPDH 和 IPO8 是最适合的 HKG。因此，我们的研究结果表明，在癌症研究中精确归一化基因表达水平时，使用正确的 HKG 至关重要，因为一种适合一种细胞类型的 HKG 不一定适合另一种细胞类型。
