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

摘要: The aim of our work was to show how a chosen normal-isation strategy can affect the outcome quantitative gene expression studies. As an example, we analysed three genes known be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: straightforward normalisation against single reference gene, GAPDH, 2−ΔΔCt algorithm more complex factor calculated from raw four previously validated genes. We found that strategies revealed contradicting results: normalising set upregulation interest in post-mortem tissue samples (cardiac muscle, skeletal muscle brain) conditions. Interestingly, statistically significant difference relative transcript abundance cardiac between donors who died asphyxia versus death. Normalisation GAPDH alone no but, some instances, downregulation interest. To further analyse this discrepancy, stability all used reassessed very low originate co-regulation concluded is not suitable for analysis hypoxia validation crucial step generating biologically meaningful data.