International Journal of Legal Medicine

, Volume 127, Issue 2, pp 335–344

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

  • Antje Huth
  • Benedikt Vennemann
  • Tony Fracasso
  • Sabine Lutz-Bonengel
  • Marielle Vennemann
Original Article

DOI: 10.1007/s00414-012-0787-2

Cite this article as:
Huth, A., Vennemann, B., Fracasso, T. et al. Int J Legal Med (2013) 127: 335. doi:10.1007/s00414-012-0787-2


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.


Post-mortem tissueRT-qPCRNormalisationHypoxiaGene expression

Supplementary material

414_2012_787_MOESM1_ESM.xlsx (13 kb)
Table S1Detailed information about donors from which samples were obtained. Bars (—) indicate unavailable information. Asterisks (*) indicate the two cases in which no brain tissue was available (XLSX 13 kb)
414_2012_787_MOESM2_ESM.xls (16 kb)
Table S2A summary of all available assay information. Assays were purchased as ready-made assays from Life Technologies and validated before use. Assay efficiencies and amplification rates are within the acceptable range suggested previously [1] (XLS 15 kb)
414_2012_787_MOESM3_ESM.pdf (71 kb)
Table S3Further validation results for the GOI assays using an artificially degraded commercially obtained RNA sample to calculate the influence of degradation on the results. The highest Cq shift was observed for HIF1A in skeletal muscle. Most Cq shifts are below 1 cycle and thus are well within the normal reaction variations (PDF 71 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Antje Huth
    • 1
    • 2
  • Benedikt Vennemann
    • 3
  • Tony Fracasso
    • 4
    • 5
  • Sabine Lutz-Bonengel
    • 1
  • Marielle Vennemann
    • 6
  1. 1.Institute of Legal MedicineFreiburg University Medical CenterFreiburgGermany
  2. 2.Eurofins MedigenomixEbersbergGermany
  3. 3.Institute for Forensic Medicine OldenburgMedical School HannoverOldenburgGermany
  4. 4.University Center of Legal MedicineUniversity of GenevaGenevaSwitzerland
  5. 5.Institute of Legal MedicineUniversity Hospital MünsterMünsterGermany
  6. 6.Institute for Forensic MedicineMedical School HannoverHannoverGermany