Holmgren Gustav, Ghosheh Nidal, Zeng Xianmin, Bogestål Yalda, Sartipy Peter, Synnergren Jane
Systems Biology Research Center, University of Skövde, Skövde, Sweden; Department of Clinical Chemistry/Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden;
Buck Institute for Research on Aging, Buck Institute, Novato, California; and.
Physiol Genomics. 2015 Jun;47(6):232-9. doi: 10.1152/physiolgenomics.00130.2014. Epub 2015 Apr 7.
Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs.
参考基因，通常被称为管家基因（HKGs），常被用于标准化基因表达数据，其依据的假设是它们在细胞中以恒定水平表达。然而，多项研究表明，不同细胞类型中HKGs的基因表达水平可能存在很大差异。在之前的一项研究中，我们利用经历自发分化的人类胚胎干细胞（hESCs），观察到常用HKG的表达变化程度使得它们在那些实验条件下不适宜用作参考基因。在此，我们展示了一项关于人类多能干细胞（hPSC）中HKG特征的大幅扩展研究，包括来自hESC和人类诱导多能干细胞的九个全局基因表达数据集，这些数据集是在向内胚层、中胚层和外胚层衍生物的定向分化过程中获得的。我们编制了稳定表达基因集，并鉴定出少数几个基因（如EID2、ZNF324B、CAPN10和RABEP2）作为在所有细胞系和实验条件下hPSC中普遍适用的参考基因。通过逆转录定量PCR分析证实了基因表达谱的稳定性。综上所述，当前结果表明分化中的hPSC具有独特的HKG特征，这在某些方面与体细胞类型不同，并强调了在实际使用的实验设置下验证参考基因稳定性的必要性。此外，本研究中鉴定出的新型假定HKG可优先用于标准化从分化中的hPSC获得的基因表达数据。
