I was trying to delete the expression of a gene with shRNA. Western blot has shown no results. Does somebody has a hypothesis of why this happened?
Low transfection efficiency is a common cause of poor silencing results. Try using a transfection reagent specific to your cell line (https://altogen.com/products-index/) and use a GFP marker as Anand recommended. Use the GFP signal to determine transfection efficiency, have positive and negative controls for your protein, and make sure that your antibody works. Westerns can have their own procedural issues, but GFP expression (even qualitatively) will help you pinpoint any procedural errors.
Dear Theo,
Please see the attached paper on the design and delivery of shRNA - I hope this will be helpful.
Best wishes,
Roland.
I think Roland added a very good reference. In addition, some proteins could be very stable. You need to perform multiple rounds of knockdowns by transfection of the shRNAs or under prolonged induction of the stably expressed shRNAs in order to see a reduction of the protein levels.
Very common.
1) Check shRNA clone, sequence, targetting area carefully again.
2) Protein is super stable. So, generate stable cells by adding selection drug.
3) Transfection efficiency is low. Use GFP marker.
Use + and - controls.
4) Use same sequence and do siRNA transfection, check if KD works.
5) Do RT PCR. May be RNA is gone done, but protein is long lived.
Good luck !
Low transfection efficiency is a common cause of poor silencing results. Try using a transfection reagent specific to your cell line (https://altogen.com/products-index/) and use a GFP marker as Anand recommended. Use the GFP signal to determine transfection efficiency, have positive and negative controls for your protein, and make sure that your antibody works. Westerns can have their own procedural issues, but GFP expression (even qualitatively) will help you pinpoint any procedural errors.
I'm facing the same problem. I used two shRNA sequences that have been validated in the sigma web site. Used lentivirus to do the transfection, selected stable cell line in 1ug/ml puromycin, and monitered with GFP. After 72h the protein level showed no change. The GFP level is different among the cells and I'm planning to selet for the brightest cells by FACS, increase the puromycin concentration and do the RT-PCR. Hope this will work! Is there any other suggestions?@ Michael Davis @ Roland Hjerpe @ Hua Xiao @Anand Patwardhan
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