I am using growth factor reduced basement membrane extract ( 10mg/ml Matrigel from ATCC) to compare 2 conditioned media (CM) from HUVEC transfected with a siRNA control negative or siRNA against one protein of interest. CMs are collected and freeze in -80°C and cells are using to isolate RNA to do qPCR. The levels of mRNA VEGF and TGFb are higher in cells transfected with siRNA against the protein of our study, compared to siRNA control. But when I do angiogenesis assay I do not observe difference in the tube formation (being same in both). Could is possible these factors could be being degraded in the CM o affected be other proteins?
After how many hours you attempted to take the pics of in vitro angiogenesis assay. According to my knowledge, you can try different time points to analyse results. If you will not get any difference at any time point, then it may try to do western blot for vegf in media after concentrating it.
I was following it during 24hrs without changes. Thanks for your comment. I will do it!. Do you know if vegf can be degradeted by proteases easily?.
As per my knowledge, tubes start disintegrating after 8 hours. You should try shorter time points like 2 hrs, 4 hrs, 8 hours. You can check regarding degradation of VEGF by proteases in below mentioned article.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977708/
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