how to evaluate the siRNA silence efficiency? - western or rtPCR (Feb/11/2007 )
I was working on the siRNA expression vector for about six month.Now I had a problem about efficiency of the silence.how to estimate the ratio?how to choose the methods to evaluate ?Most institutes apply the western blot or rt-PCR.But my problem are that:1.which method is better to estmate ,2.how much the siRNA expression vector silence the target gene can identify the siRNA useful.
RT-PCR will be my choice to prove knockdown (more sensitive and quantitative). But I wouldnt mind western if the knockdown is obvious.
I apologize for I donot understand what exactly do you need by the second question.
I would think Western is better if you want to be more certain of silencing effects, it's after all the protein that will have effects downstream
However, realtime is much simpler when you are optimising and takes less time
Both methods are acceptable if you are thinking of publications
Did you mean how mcuh %of silencing will mean the siRNA is working properly?
Generally, at least a 70% knockdown is acceptable, while it is also possible to go down to 90%
Hope it helps
thanks to the up ladder
I have read some paper:they found the mRNA silence efficiency is about 50%,but the western results disclose the protein silence efficiency is 90% or more.So I have the questions:which is the best way to evalute the silence.
Like sharonpek said, realtime PCR remains the best way to evaluate the silence efficiency. Because it uses fluorecence as an indicator of the amount of dsDNA, it is much more precise than the western blot.
But you can surely show the knockdown with western blot. Just quantify with Q-PCR
i have a question which is similar to the ones we have here, we all know that its proven that 90% of all rna in e.coli is not translated into protein and hence is doing a q-rtpcr effective to detect silencing, i mean the rna could be present but it might not be translated into the protein, so doing a western blot will only take into account the protein that is which causes all the changes.
also isn't ELISA an option that can be considered instead of western, its the same technique except that the blotting part is not required. can this be used instead of doing a western to analyze silencing.
The nice feature of Westerns is that you get mass information. That helps support the hypothesis that your antibody is sufficiently specific for the analyte of interest. Once the behavior of the antibody has been well-characterized in the system of interest, switching to ELISA can speed up the process. For a paper in an unfamiliar system, I'd want to see a Western.