title: Reference genes for expression studies in hypoxia and hyperglycemia models in human umbilical vein endothelial cells.
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), β-glucuronidase (GUSB), and β-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.
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle NE2 4HH, United Kingdom Biochemistry Department, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia Center of Excellence in Genomic Medicine Research, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia.
authors.forename: Sherin
authors.identifier: http://orcid.org/0000-0003-1580-0409
authors.initials: S
authors.lastname: Bakhashab
authors.affiliation: Biochemistry Department, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors.forename: Sahira
authors.initials: S
authors.lastname: Lary
authors.affiliation: Center of Excellence in Genomic Medicine Research, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia.
authors.forename: Farid
authors.initials: F
authors.lastname: Ahmed
authors.affiliation: Center of Excellence in Genomic Medicine Research, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia.
authors.forename: Hans-Juergen
authors.initials: HJ
authors.lastname: Schulten
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle NE2 4HH, United Kingdom.
authors.forename: Ayat
authors.initials: A
authors.lastname: Bashir
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle NE2 4HH, United Kingdom.
authors.forename: Fahad W
authors.initials: FW
authors.lastname: Ahmed
authors.affiliation: Biochemistry Department, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors.forename: Abdulrahman L
authors.initials: AL
authors.lastname: Al-Malki
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medicine, King Abdulaziz University, P.O. Box 80215, Jeddah 21589, Saudi Arabia.
authors.forename: Hasan S
authors.initials: HS
authors.lastname: Jamal
authors.affiliation: Center of Excellence in Genomic Medicine Research, King Abdulaziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabia.
authors.forename: Mamdooh A
authors.initials: MA
authors.lastname: Gari
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle NE2 4HH, United Kingdom Jolanta.Weaver@newcastle.ac.uk.
authors.forename: Jolanta U
authors.initials: JU
authors.lastname: Weaver
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D005993:Glycerolphosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D006943:Hyperglycemia / Q000235:genetics*; D000860:Hypoxia / Q000235:genetics*; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome
chemical_list: D000199:Actins; D015703:Antigens, CD; C068126:CD71 antigen; D011990:Receptors, Transferrin; D012269:Ribosomal Proteins; C054675:ribosomal protein P0; D005993:Glycerolphosphate Dehydrogenase; D005966:Glucuronidase
keywords: HUVEC; hyperglycemia; hypoxia; reference genes