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Reference Genes for Expression Studies in Hypoxia and Hyperglycemia Models in Human Umbilical Vein Endothelial Cells

Lookup NU author(s): Sherin Bakhashab, Dr Fahad Ahmed, Dr Jolanta Weaver

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

Human umbilical vein endothelial cell (HUVEC)-based gene expression studies performed under hypoxia and/or hyperglycemia show huge potential for modeling endothelial cell response in cardiovascular disease and diabetes. However, such studies require reference genes that are stable across the whole range of experimental conditions. These reference genes have not been comprehensively defined to date. We applied human genome-wide microarrays and quantitative real-time PCR (qRT-PCR) on RNA obtained from primary HUVEC cultures that were incubated for 24 hr either in euglycemic or in hyperglycemic conditions and then subjected to short-term CoCl2-induced hypoxia for 1, 3, or 12 hr. Using whole-transcript arrays, we selected 10 commonly used reference genes with no significant expression variation across eight different conditions. These genes were ranked using NormFinder software according to their stability values. Consequently, five genes were selected for validation by qRT-PCR. These were ribosomal protein large P0 (RPLP0), transferrin receptor (TFRC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), and beta-actin (ACTB). All five genes displayed stable expression under hyperglycemia. However, only RPLP0 and TFRC genes were stable under hypoxia up to 12 hr. Under hyperglycemia combined with hypoxia up to 12 hr, the expression of RPLP0, TFRC, GUSB, and ACTB genes remained unchanged. Our findings strongly confirm that RPLP0 and TFRC are the most suitable reference genes for HUVEC gene expression experiments subjected to hypoxia and/or hyperglycemia for the given experimental conditions. We provide further evidence that even commonly known references genes require experimental validation for all conditions involved.


Publication metadata

Author(s): Bakhashab S, Lary S, Ahmed F, Schulten HJ, Bashir A, Ahmed FW, Al-Malki AL, Jamal HS, Gari MA, Weaver JU

Publication type: Article

Publication status: Published

Journal: G3: Genes Genomes Genetics

Year: 2014

Volume: 4

Issue: 11

Pages: 2159-2165

Print publication date: 01/11/2014

Online publication date: 05/09/2014

Acceptance date: 01/09/2014

Date deposited: 16/11/2015

ISSN (electronic): 2160-1836

Publisher: Genetics Society of America

URL: http://dx.doi.org/10.1534/g3.114.013102

DOI: 10.1534/g3.114.013102

PubMed id: 25193495


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