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Lookup NU author(s): Sherin Bakhashab, Dr Fahad Ahmed, Dr Jolanta Weaver
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
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.
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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