I will perform a apoptosis evaluation in cell lines using flow cytometry after siRNA transfection, and I want to evaluate the levels of BAX and BCL-2 to complement the experiment. However, I can only do a qPCR of both transcript, but not a western blotting. Can qPCR be used in this case, or it would be only informative if I measure the protein levels?
I recommend you to use Caspase3 activation as an indicator of apoptosis. ((western blotting technique)).
good luck .
Hello Alexandre Luiz Korte de Azevedo
Yes, qPCR can be used. It would be the most direct detection method to measure siRNA-mediated target knockdown. However, while using qPCR, the design of primers is an important consideration since using a primer set targeted to the wrong region may result in false negative results.
I would recommend that you should also perform Western Blot if you have the facility since it will show whether it is having the desired effect on functional protein as well. The qPCR results will show that the mRNA level of each gene is significantly lowered in the transfected cells than control cells. The western blot results can be used to correlate with the qPCR results where the protein band intensity reduction may be seen in siRNA-treated cells.
In this way, you could have valuable insights from protein as well that you will miss if you decide to just look at the transcripts alone.
Best.
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