Hello, I have silenced some genes which is upper than MAPK. Now I want to get the iNOS change and NO change. My NO is more down-regulated than siNC about half a fold, however, my iNOS is more up-regulated than siNC. The siRNA efficiency of my genes is about 30%.
What is the problem with my assay?
iNOS may generate both superoxide anion and NO. I guess the remaining one you have makes O2*- instead. This hypothesis may be tested by measuring O2*- and H2O2 using fluorescent dyes/luminiscence or reduced-to-oxidised glutathione ratio.
Hernansanz-Agustín P et al. Acute hypoxia produces a superoxide burst in cells.Free Radic Biol Med. 2014 Jun;71:146-56. doi: 10.1016/j.freeradbiomed.2014.03.011. Epub 2014 Mar 15.
Mihov D, et al.,.Erythropoietin activates nitric oxide synthase in murine erythrocytes. Am J Physiol Cell Physiol. 2009 Aug;297(2):C378-88.
Your cells or tissues may be depleted of tetrahydrobiopterin which will uncouple iNOS activity to increase superoxide production.
I agree with Kirkwood. Please check the H4-bioterin level , measure GTP -cyclo hydrolase 1 level as well examine if sepiaterin supplement to cell culture can increase the NO level. Also check dimer -monomer ratio of iNOS protein by low temperature SDS gel in absence of thiol( reducing agent)( treated vs untreated control )---best of luck---Dipak Ghosh
If you are simply measuring nitrite as the metric for NO production, you could be getting the discrepancies (through mechanisms noted above) that leads to alternative nitrogen oxides as well as a diversion to superoxide. If you follow nitrated tyrosine as well you may get a clearer picture.
Besides the siRNA efficiency remember that you have different forms of nitric oxide synthases, such as iNOS eNOS, nNOS, etc, regarding the tissue. Just confirm this won´t be a relevant aspect.
I agree with most others. Besides you say there is reduction in NO, which method you are using? As Antonio said its worth looking for eNOS and nNOs. Better look at Superoxide levels first.
Question is that any cells has all of 3 genes for NOS (neuronal, inducible and endothelial specific). Therefore it is almost impossible to make verification. In addition detect NO cannot be as a specific markers to the any specific isoforms. You should do immunocytochemistry and gene analysis and then you can compare to the amount of NO which you measured by ELISA or any other techniques.
Yeah you may well be getting uncoupled NO synthesis accompanied by superoxide formation if there is a deficiency of substrate or cofactors as pointed out by most. But all depends on how you are measuring the NO. If you are doing an in-situ Griess assay you may perhaps encounter the type of problem you are facing. In order to figure out whether this mismatch is because of substrate (arginine) or cofactor (tetrahydrobiopterin, FMN and FAD) limitation in your assay system, you may purify the iNOS and conduct a standard NOS activity assay using standard protocols and assay mix.. Your specific activity values should match the expression levels of iNOS if its in active dimeric form. If it still doesn't match, check the dimer monomer content of your iNOS protein as suggested by Dr. Ghosh either by gel filtration or cold non-reducing gel electrophoresis without adding excess tetrahydrobiopterin or arginine to the protein (as that may influence the real dimer content). Also spectrophotometrically check the P450 level of the NOS heme as this is the fundamental determinant of the catalytic efficacy of the NOS protein. If you manage to get and use some iNOS positive controls along with your experimental iNOS protein you would get a clearer indication of whether your NOS protein is at all catalytically defective should you continue to see that the activity levels are in mismatch with the levels of the generated iNOS proteins.
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