Hi.. I am using mouse cell lines and shRNAs for my knockdown and I have obtained stables for my candidate genes. I performed qPCR and there was no Cp value for KD samples. I did it thrice and every time I got the same result [No Cp value]. My internal control for the same sample however gave the expected Cp value. I also checked my qPCR products by gel electrophoresis and the expected band was obtained for those sample that had a Cp value. Melting curves also looked good. Should I rely on these results? Because I have never seen anyone having a complete knockdown!!
It will be good to do a western blot for these proteins to confidently conclude a complete knockdown.
Hi Deepak.. Thanks for the reply. Yes I will be doing a western blot but before ordering them I just want a suggestion on the probability of a complete knockdown.
Hi Onur.. I did viral transduction and got an efficiency of 40-50%
Complete knockdown it's almost impossible, If you want a 100% loss of translation then you should consider a knockout model.
You're lucky reaching a 20-30% residual expression through qRT-PCR analysis, usually a 40-50% is a common outcome. Don't be afraid if you have a stronger effect and also if you have a less stronger effect.
Different factors interplay a role in knockdown efficiency at the transcript level, for example the saturation of DICER/RISC complexes which are the limiting steps in interference RNAs processing, the clearance of unprocessed interference RNAs by cells themselves and also the intrinsic stability of interference RNAs and last but not least the basic level of expression of the target (that mean the mRNA abundance).
Not only this but the final outcome of knockdown is always related with the protein turnover. You can observe a 70% loss of expression through qRT-PCR with a blotting outcome of less than 30%. The more the protein is stable and durable the more is difficult to have a knockdown final effect. On the other side a fast-turnover protein will increase the final effect of KD.
Indeed as suggested by Deepak Kanojia, a wester blotting analysis will be good at this purpose.
@Paolo thanks for the answer. Thanks for the detailed explanation. I was also wondering if the number of target molecules would have an effect. Like if the expression is already on the moderate side, then knocking it down should reduce its expression further and hence the number of target molecules.. Probably I should try a different dilutin range of cDNA just to make sure I have some target molecules in knockdown.
If you have a non-detectable Ct value through qRT-PCR in you knockdown cells (primers are "quantitative" enough and that makes qRT-PCR reliable) and your target is non super-abundantly expressed then probably your final effect will be dependent initially more on protein turnover rather than RNAi machinery. You just should perform a time course western blotting analysis to ensure the proper protein reduction.
good luck!
@Paola.. Sorry for getting back late.. As I said before, I actually tried the qRT changing the dilution range and it worked perfectly.. I could see the knockdown for both my candidate genes.!! Let me see how the protein levels look like!! Thank you..
never saw a complete KD with si/shRNA. can be 99% in some cases though- eg we have a super potent siRNA for GAPDH
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