Moein Shiva, Javanmard Shaghayegh Haghjooy, Abedi Maryam, Izadpanahi Mohammad Hosein, Gheisari Yousof
Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran.
Applied Physiology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.
Adv Biomed Res. 2017 Feb 22;6:15. doi: 10.4103/2277-9175.200790. eCollection 2017.
Selection of stably expressing housekeeping genes (HKGs) is a crucial step in gene expression analysis. However, there are no universal HKGs for all experiments, and they should be determined by each biologic condition. The aim of this study was to detect appropriate HKGs for kidney cells cultured in long-term hypoxia.
Based on a screening step using a microarray data available from gene expression omnibus database, a set of candidate HKGs were chosen to be assessed in human kidney cells cultured in hypoxic or normoxic conditions for about 2 weeks in a time course manner. The stability of gene expression was assessed by refFinder, a web-based tool that integrates four computational programs (geNorm, Normfinder, BestKeeper, and the comparative ΔΔCt method).
GAPDH and ACTB were the most stable genes in hypoxia treated cells whereas, B2M and ACTB were the best HKGs in cells cultured in normoxia. When both hypoxia and normoxia treated cells from all time points were evaluated together, GAPDH and ACTB equally showed the most stability.
As in relative quantification of real-time polymerase chain reaction data, the same HKGs should be selected for all groups, we believe that GAPDH and ACTB are suitable HKGs for studies on the effect of hypoxia on cultured kidney cells.
选择稳定表达的管家基因（HKGs）是基因表达分析中的关键步骤。然而，不存在适用于所有实验的通用HKGs，它们应由每种生物学条件来确定。本研究的目的是检测在长期缺氧条件下培养的肾细胞的合适HKGs。
基于使用从基因表达综合数据库获得的微阵列数据的筛选步骤，选择一组候选HKGs，以时间进程方式在缺氧或常氧条件下培养约2周的人肾细胞中进行评估。通过refFinder评估基因表达的稳定性，refFinder是一种基于网络的工具，整合了四个计算程序（geNorm、Normfinder、BestKeeper和比较ΔΔCt方法）。
在缺氧处理的细胞中，GAPDH和ACTB是最稳定的基因，而在常氧培养的细胞中，B2M和ACTB是最佳的HKGs。当对所有时间点的缺氧和常氧处理细胞一起评估时，GAPDH和ACTB同样显示出最高的稳定性。
由于在实时聚合酶链反应数据的相对定量中，所有组应选择相同的HKGs，我们认为GAPDH和ACTB是研究缺氧对培养肾细胞影响的合适HKGs。
