I'm going to expose cells to hypoxic conditions, in order to establish the function of a synthetic promoter containing HRE's. For control sake, I want to see if cells actually developed hypoxic stress.
Does any one knows a good reliable assay or biomarker for cellular hypoxic stress? Preferably something you can use in a sorter, but RT-PCR and other methods are also welcome.
Thanks in advance
The simplest method to find out the level of hypoxia is to measure HIF1 alfa . You can do it by RT-PCR.
easy way to would be to a luciferase assay I have attached one of my publication
Article Targeted delivery of the hydroxylase inhibitor DMOG provides...
HIF1alpha is not neccesarily regulated on mRNA level, so RT-PCR can be misleading. It would be better to either show the post-translational modifications (western blots) or to show its translocation to the nucleus (nuclear extract western blots). However, it might be more convenient to analyze the expression of some HIF traget genes. This review
http://faculty.sites.uci.edu/razorenova-lab/files/2013/04/HIF-review-Semenza.pdf
gives a good overview.
I agree with Jochen above, qPCR analysis of HIFa isoforms is very misleading, these will not necessarily change under hypoxic conditions. Essentially you will see an increase in the stability of the protein, as the hydroxylation of the proline residues decrease. Western blots for HIF1a or HIF2a expression, or look for target gene expression, Ladh, VEGF, PDK1, SERPIN1a are a few suggestions. You also need to consider the length of the hypoxic time course as the target expression can vary depending on which of the HIFa isoforms is stabilised. HIF1 stability occurs very rapidly, HIF2a requires a little more time to become fully functional 10-12hr.
- Badges
- Science topic