Ear Science Institute Australia, Nedlands, Western Australia, Australia.
Ear Sciences Centre, The University of Western Australia, Nedlands, Western Australia, Australia.
BMC Mol Cell Biol. 2023 Apr 17;24(1):16. doi: 10.1186/s12860-023-00475-4.
Hypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of some commonly used housekeeping genes, such as GAPDH and PGK1, can confound the relative gene expression analysis. Thus, there is ongoing debate as to which housekeeping gene is appropriate for studies investigating hypoxia-induced cell responses. Specifically, there is still contradicting information for which housekeeping genes are stable in hypoxia cultures of mesenchymal stem cells. In this study, candidate housekeeping genes curated from the literature were matched to RNAseq data of normoxic and hypoxic human adipose-derived stem cell cultures to determine if gene expression was modulated by hypoxia or not. Expression levels of selected candidates were used to calculate coefficient of variation. Then, accounting for the mean coefficient of variation, and normalised log twofold change, genes were ranked and shortlisted, before validating with qRT-PCR. Housekeeping gene suitability were then determined using GeNorm, NormFinder, BestKeeper, comparative[Formula: see text], RefFinder, and the Livak method.
Gene expression levels of 78 candidate genes identified in the literature were analysed in the RNAseq dataset generated from hADSC cultured under Nx and Hx conditions. From the dataset, 15 candidates with coefficient of variation ≤ 0.15 were identified, where differential expression analysis results further shortlisted 8 genes with least variation in expression levels. The top 4 housekeeping gene candidates, ALAS1, RRP1, GUSB, and POLR2B, were chosen for qRT-PCR validation. Additionally, 18S, a ribosomal RNA commonly used as housekeeping gene but not detected in the RNAseq method, was added to the list of housekeeping gene candidates to validate. From qRT-PCR results, 18S and RRP1 were determined to be stably expressed in cells cultured under hypoxic conditions.
We have demonstrated that 18S and RRP1 are suitable housekeeping genes for use in hypoxia studies with human adipose-derived stem cell and should be used in combination. Additionally, these data shown that the commonly used GAPDH and PGK1 are not suitable housekeeping genes for investigations into the effect of hypoxia in human adipose-derived stem cell.
缺氧培养条件已被用于研究许多疾病模型中缺氧对基因表达的影响。然而，一些常用管家基因启动子区域中的缺氧反应元件，如 GAPDH 和 PGK1，可能会混淆相对基因表达分析。因此，关于哪种管家基因适合研究缺氧诱导的细胞反应，一直存在争议。具体来说，对于骨髓间充质干细胞在缺氧培养中哪些管家基因稳定，仍存在相互矛盾的信息。在这项研究中，从文献中挑选出候选管家基因，并与常氧和缺氧的人脂肪来源干细胞培养的 RNAseq 数据进行匹配，以确定基因表达是否受到缺氧的调节。选择的候选基因的表达水平用于计算变异系数。然后，考虑到平均变异系数和归一化的两倍变化对数，对基因进行排名和筛选，最后用 qRT-PCR 进行验证。然后使用 GeNorm、NormFinder、BestKeeper、comparative[Formula: see text]、RefFinder 和 Livak 方法来确定管家基因的适用性。
从在 Nx 和 Hx 条件下培养的 hADSC 的 RNAseq 数据集分析了文献中鉴定的 78 个候选基因的表达水平。从数据集中确定了 15 个变异系数≤0.15 的候选基因，差异表达分析结果进一步将 8 个表达水平变化最小的基因进行了筛选。前 4 个管家基因候选基因 ALAS1、RRP1、GUSB 和 POLR2B，被选为 qRT-PCR 验证的候选基因。此外，18S，一种常用于作为管家基因但在 RNAseq 方法中未检测到的核糖体 RNA，也被添加到管家基因候选列表中进行验证。从 qRT-PCR 结果来看，18S 和 RRP1 被确定在缺氧条件下培养的细胞中稳定表达。
我们已经证明 18S 和 RRP1 是适用于人脂肪来源干细胞缺氧研究的管家基因，应联合使用。此外，这些数据表明，常用的 GAPDH 和 PGK1 不适合用于研究缺氧对人脂肪来源干细胞的影响。
