Hello everyone,
I am working on MDAMB231 cell line in which I have to knock down molecule of 25-37Kda .I tried both siRNA(Dharmafect mediated,Invitrogen) and shRNA(LTX,PLUS mediated,Invitrogen) with different conditions and for different time period, but unable to get knock down(neither at mRNA level nor at protein level).Can someone help me in troubleshooting it?
First off, has siRNA or shRNA knockdown of this particular protein been shown in the literature for your cell line? If not, the troubleshooting could be quite extensive. If so, make sure you are following existing protocols very closely, as the timing for knockdown (both in terms of how long to incubate with the RNA and how long to wait before analysis) can vary wildly for different cell types and proteins.
On another note, I presume the protein of interest isn't vital. If it's essential, knockdown may either be cytotoxic or other mechanisms may be kicking in to boost production.
Thank you Ross.Though I tried following the same protocol which worked always in our lab and which is reported in literature also, but couldn't get the desired result.I wish I could resolve the issue at the earliest.
Hi Sameena
- check the quality of siRNA in gel - it may be of less concentration or degradated
- check transfection efficiency via GFP and silencing efficiency via anti-GFP siRNA
- get some additional siRNA either at CAP, PolyA region or at the middle
good luck
Thank you so much Andrey. I wll try the second suggestion which you have given.Hope It work for me and I can get desired result.
Good luck
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