PMID- 25193495
OWN - NLM
STAT- MEDLINE
DCOM- 20150723
LR  - 20220317
IS  - 2160-1836 (Electronic)
IS  - 2160-1836 (Linking)
VI  - 4
IP  - 11
DP  - 2014 Sep 5
TI  - Reference genes for expression studies in hypoxia and hyperglycemia models in 
      human umbilical vein endothelial cells.
PG  - 2159-65
LID - 10.1534/g3.114.013102 [doi]
AB  - 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.
CI  - Copyright (c) 2014 Bakhashab et al.
FAU - Bakhashab, Sherin
AU  - Bakhashab S
AUID- ORCID: 0000-0003-1580-0409
AD  - 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.
FAU - Lary, Sahira
AU  - Lary S
AD  - Biochemistry Department, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, 
      Saudi Arabia.
FAU - Ahmed, Farid
AU  - Ahmed F
AD  - Center of Excellence in Genomic Medicine Research, King Abdulaziz University, 
      P.O. Box 80216, Jeddah 21589, Saudi Arabia.
FAU - Schulten, Hans-Juergen
AU  - Schulten HJ
AD  - Center of Excellence in Genomic Medicine Research, King Abdulaziz University, 
      P.O. Box 80216, Jeddah 21589, Saudi Arabia.
FAU - Bashir, Ayat
AU  - Bashir A
AD  - Institute of Cellular Medicine, Newcastle University, Newcastle NE2 4HH, United 
      Kingdom.
FAU - Ahmed, Fahad W
AU  - Ahmed FW
AD  - Institute of Cellular Medicine, Newcastle University, Newcastle NE2 4HH, United 
      Kingdom.
FAU - Al-Malki, Abdulrahman L
AU  - Al-Malki AL
AD  - Biochemistry Department, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, 
      Saudi Arabia.
FAU - Jamal, Hasan S
AU  - Jamal HS
AD  - Department of Obstetrics and Gynaecology, Faculty of Medicine, King Abdulaziz 
      University, P.O. Box 80215, Jeddah 21589, Saudi Arabia.
FAU - Gari, Mamdooh A
AU  - Gari MA
AD  - Center of Excellence in Genomic Medicine Research, King Abdulaziz University, 
      P.O. Box 80216, Jeddah 21589, Saudi Arabia.
FAU - Weaver, Jolanta U
AU  - Weaver JU
AD  - Institute of Cellular Medicine, Newcastle University, Newcastle NE2 4HH, United 
      Kingdom Jolanta.Weaver@newcastle.ac.uk.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20140905
PL  - England
TA  - G3 (Bethesda)
JT  - G3 (Bethesda, Md.)
JID - 101566598
RN  - 0 (Actins)
RN  - 0 (Antigens, CD)
RN  - 0 (CD71 antigen)
RN  - 0 (Receptors, Transferrin)
RN  - 0 (Ribosomal Proteins)
RN  - 0 (ribosomal protein P0)
RN  - EC 1.1.- (Glycerolphosphate Dehydrogenase)
RN  - EC 3.2.1.31 (Glucuronidase)
SB  - IM
MH  - Actins/genetics/metabolism
MH  - Antigens, CD/genetics/metabolism
MH  - Gene Expression Profiling/methods/*standards
MH  - Glucuronidase/genetics/metabolism
MH  - Glycerolphosphate Dehydrogenase/genetics/metabolism
MH  - Human Umbilical Vein Endothelial Cells/*metabolism
MH  - Humans
MH  - Hyperglycemia/*genetics
MH  - Hypoxia/*genetics
MH  - Receptors, Transferrin/genetics/metabolism
MH  - Ribosomal Proteins/genetics/metabolism
MH  - Transcriptome
PMC - PMC4232541
OTO - NOTNLM
OT  - HUVEC
OT  - hyperglycemia
OT  - hypoxia
OT  - reference genes
EDAT- 2014/09/07 06:00
MHDA- 2015/07/24 06:00
PMCR- 2014/09/05
CRDT- 2014/09/07 06:00
PHST- 2014/09/07 06:00 [entrez]
PHST- 2014/09/07 06:00 [pubmed]
PHST- 2015/07/24 06:00 [medline]
PHST- 2014/09/05 00:00 [pmc-release]
AID - g3.114.013102 [pii]
AID - GGG_013102 [pii]
AID - 10.1534/g3.114.013102 [doi]
PST - epublish
SO  - G3 (Bethesda). 2014 Sep 5;4(11):2159-65. doi: 10.1534/g3.114.013102.