Identification of Appropriate Housekeeping Genes for Gene Expression Analysis in Long-term Hypoxia-treated Kidney Cells

Moein, Shiva; Haghjooy Javanmard, Shaghayegh; Abedi, Maryam; Hosein Izadpanahi, Mohammad; Gheisari, Yousof

Identification of Appropriate Housekeeping Genes for Gene Expression Analysis in Long-term Hypoxia-treated Kidney Cells

Document Type : Original Article

Authors

¹ Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran

² Applied Physiology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

³ Department of Urology, Isfahan University of Medical Sciences, Isfahan, Iran

⁴ Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan; Regenerative Medicine Lab, Isfahan Kidney Diseases Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: 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. Materials and Methods: 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). Results: 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. Conclusion: 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.

Keywords

References

1.	Fraisl P, Aragonés J, Carmeliet P. Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory disease. Nat Rev Drug Discov 2009;8:139-52.
2.	Higgins DF, Kimura K, Iwano M, Haase VH. Hypoxia-inducible factor signaling in the development of tissue fibrosis. Cell Cycle 2008;7:1128-32.
3.	Fine LG, Bandyopadhay D, Norman JT. Is there a common mechanism for the progression of different types of renal diseases other than proteinuria? Towards the unifying theme of chronic hypoxia. Kidney Int Suppl 2000;75:S22-6.
4.	Fine LG, Norman JT. Chronic hypoxia as a mechanism of progression of chronic kidney diseases: From hypothesis to novel therapeutics. Kidney Int 2008;74:867-72.
5.	Legrand M, Mik EG, Johannes T, Payen D, Ince C. Renal hypoxia and dysoxia after reperfusion of the ischemic kidney. Mol Med 2008;14:502-16.
6.	Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc 2006;1:1559-82.
7.	Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: Validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 2000;46:69-81.
8.	de Kok JB, Roelofs RW, Giesendorf BA, Pennings JL, Waas ET, Feuth T, et al. Normalization of gene expression measurements in tumor tissues: Comparison of 13 endogenous control genes. Lab Invest 2005;85:154-9.
9.	Liu W, Shen SM, Zhao XY, Chen GQ. Targeted genes and interacting proteins of hypoxia inducible factor-1. Int J Biochem Mol Biol 2012;3:165-78.
10.	Costello CM, Howell K, Cahill E, McBryan J, Konigshoff M, Eickelberg O, et al. Lung-selective gene responses to alveolar hypoxia: Potential role for the bone morphogenetic antagonist gremlin in pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2008;295:L272-84.
11.	Nagelkerke A, Mujcic H, Wouters B, Span PN. 18S is an appropriate housekeeping gene for in vitro hypoxia experiments. Br J Cancer 2010;103:590.
12.	Foldager CB, Munir S, Ulrik-Vinther M, Søballe K, Bünger C, Lind M. Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes. BMC Mol Biol 2009;10:94.
13.	Bakhashab S, Lary S, Ahmed F, Schulten HJ, Bashir A, Ahmed FW, et al. Reference genes for expression studies in hypoxia and hyperglycemia models in human umbilical vein endothelial cells. G3 (Bethesda, Md). 2014;4:2159-65.
14.	Caradec J, Sirab N, Keumeugni C, Moutereau S, Chimingqi M, Matar C, et al. 'Desperate house genes': The dramatic example of hypoxia. Br J Cancer 2010;102:1037-43.
15.	Tan SC, Carr CA, Yeoh KK, Schofield CJ, Davies KE, Clarke K. Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors. Mol Biol Rep 2012;39:4857-67.

Advanced Biomedical Research