Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation.

Details

Serval ID
serval:BIB_E734FFD7D641
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation.
Journal
International Journal of Legal Medicine
Author(s)
Huth A., Vennemann B., Fracasso T., Lutz-Bonengel S., Vennemann M.
ISSN
1437-1596 (Electronic)
ISSN-L
0937-9827
Publication state
Published
Issued date
2013
Peer-reviewed
Oui
Volume
127
Number
2
Pages
335-344
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't Publication Status: ppublish
Abstract
The aim of our work was to show how a chosen normal-isation strategy can affect the outcome of quantitative gene expression studies. As an example, we analysed the expression of three genes known to be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: a straightforward normalisation against a single reference gene, GAPDH, using the 2(-ΔΔCt) algorithm and a more complex normalisation against a normalisation factor calculated from the quantitative raw data from four previously validated reference genes. We found that the two different normalisation strategies revealed contradicting results: normalising against a validated set of reference genes revealed an upregulation of the three genes of interest in three post-mortem tissue samples (cardiac muscle, skeletal muscle and brain) under hypoxic conditions. Interestingly, we found a statistically significant difference in the relative transcript abundance of VEGF in cardiac muscle between donors who died of asphyxia versus donors who died from cardiac death. Normalisation against GAPDH alone revealed no upregulation but, in some instances, a downregulation of the genes of interest. To further analyse this discrepancy, the stability of all reference genes used were reassessed and the very low expression stability of GAPDH was found to originate from the co-regulation of this gene under hypoxic conditions. We concluded that GAPDH is not a suitable reference gene for the quantitative analysis of gene expression in hypoxia and that validation of reference genes is a crucial step for generating biologically meaningful data.
Keywords
Adolescent, Adult, Aged, Aged, 80 and over, Anoxia/genetics, Anoxia/pathology, Brain/metabolism, Brain/pathology, Female, Gene Expression, Genes, Essential/genetics, Glucose Transporter Type 1/genetics, Glyceraldehyde-3-Phosphate Dehydrogenases/genetics, Humans, Hypoxia-Inducible Factor 1, alpha Subunit/genetics, Male, Middle Aged, Muscle, Skeletal/metabolism, Muscle, Skeletal/pathology, Myocardium/metabolism, Myocardium/pathology, Reverse Transcriptase Polymerase Chain Reaction, Up-Regulation, Vascular Endothelial Growth Factor A/genetics, Young Adult
Pubmed
Web of science
Create date
08/12/2014 17:32
Last modification date
20/08/2019 16:10
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