Selection of Reference Genes for Studies of Human Retinal Endothelial Cell Gene Expression by Reverse Transcription-Quantitative Real-Time Polymerase Chain Reaction.
Selection of Reference Genes for Studies of Human Retinal Endothelial Cell Gene Expression by Reverse Transcription-Quantitative Real-Time Polymerase Chain Reaction.
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DOI：
10.1016/j.genrep.2017.11.009
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发表时间：
2018-03
期刊：
影响因子：
1.3
通讯作者：
Smith JR
中科院分区：
文献类型：
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作者：
Appukuttan B;Ashander LM;Ma Y;Smith JR
人视网膜内皮细胞越来越多地用于视网膜血管疾病的研究。通过逆转录定量实时聚合酶链反应 (RT-qPCR) 分析基因表达对疾病相关刺激的反应很常见。然而，大多数报道的工作并未遵循 qPCR 实验发表的最低信息 (MIQE) 建议，即使用多个稳定表达的参考基因进行标准化。两种人视网膜内皮细胞系仅用培养基或含有刺激物的培养基处理，所述刺激物包括：超生理浓度的葡萄糖、二甲基草酰甘氨酸、血管内皮生长因子、肿瘤坏死因子-α、脂多糖和弓形虫速殖子。通过测量刺激相关转录物的显着增加来证实细胞的生物反应。通过设计用于检测 28 个参考基因的商业 PCR 阵列对总 RNA 进行逆转录和分析。对于每个和两个细胞系以及对于每个和所有条件，参考基因表达的稳定性是根据小于 0.2 的基因稳定性测量（M 值）和小于 0.1 的变异系数（CV 值）来判断的。参考基因表达在不同刺激和细胞系之间存在显着差异。在 27 个可检测的参考基因中，11-21 个 (41-78%) 在刺激和细胞系中保持表达稳定性。排名表明在不同条件下最稳定的参考基因存在显着的多样性，并且对于两种细胞系，在所有刺激条件下没有参考基因稳定表达。 4个内参基因在5种条件下稳定表达：HSP90AB1、IPO8、PSMC4和RPLPO。我们观察到不同刺激下以及人视网膜内皮细胞系之间参考基因表达稳定性的变化。我们的研究结果支持遵守 MIQE 关于人视网膜内皮细胞 RT-qPCR 研究标准化的建议。
Human retinal endothelial cells are employed increasingly for investigations of retinal vascular diseases. Analysis of gene expression response to disease-associated stimuli by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is common. However, most reported work does not follow the minimum information for publication of qPCR experiments (MIQE) recommendation that multiple, stably expressed reference genes be used for normalization. Two human retinal endothelial cell lines were treated with medium alone or containing stimuli that included: glucose at supraphysiological concentration, dimethyloxalyl-glycine, vascular endothelial growth factor, tumor necrosis factor-α, lipopolysaccharide and Toxoplasma gondii tachyzoites. Biological response of cells was confirmed by measuring significant increase in a stimulus-relevant transcript. Total RNA was reverse transcribed and analyzed by commercial PCR arrays designed to detect 28 reference genes. Stability of reference gene expression, for each and both cell lines, and for each and all conditions, was judged on gene-stability measure (M-value) less than 0.2 and coefficient of variation (CV-value) less than 0.1. Reference gene expression varied substantially across stimulations and between cell lines. Of 27 detectable reference genes, 11–21 (41–78%) maintained expression stability across stimuli and cell lines. Ranking indicated substantial diversity in the most stable reference genes under different conditions, and no reference gene was expressed stably under all conditions of stimulation and for both cell lines. Four reference genes were expressed stably under 5 conditions: HSP90AB1, IPO8, PSMC4 and RPLPO. We observed variation in stability of reference gene expression with different stimuli and between human retinal endothelial cell lines. Our findings support adherence to MIQE recommendations regarding normalization in RT-qPCR studies of human retinal endothelial cells.