I knocked down a gene which has been shown to promote cell proliferation. qPCR results confirmed that the gene was indeed knocked down. There was increased expression of p53 as a result, which also correlated with apoptosis. I had two negative controls which helped me confirm that cell death was as a result of knockdown, not non-specific effects. However, the western blot does not show expression of the protein in untreated cells, but expression rather goes up when I knocked down. The 1st lane is the untreated, the fourth lane shows transfection with siRNA. (The protein is about 250kD).
Probably, you should give more information on the time you do the transfection and harvesting of cells for qPCR and western blot?
Thank you King. I transfected for 48hours, treated with taxol and Camptothecin for 24 hours, then isolated proteins.
I would take a step back and just check that the siRNA can reduce your protein without the added complications of treating the cells. Unless the protein you are knocking down is only expressed under the treatment conditions? 250kD is a very large protein and may be troublesome to transfer so first be sure that the band you are looking at is the right one. Have a simple gel with 1 lane with a negative control siRNA and one lane with the specific targeting siRNA. Check with Ponceau stain that transfer was even and also probe for expression of a housekeeper that should be equal in both samples.
Aside from this, doesn't Taxol and CPT treatment induce p53 and cell death in your model system anyway, without knocking down your protein of interest?
Thank you very much Amanda. The idea of knocking down the protein (which is pro-survival in many cases) is to improve upon apoptosis. I did an overnight transfer at 4 degrees and confirmed transfer with Ponceau stain. Yes. Taxol and CPT induce cell death. I just did not expect to find the protein after knockdown of the mRNA. What confuses me the most is that expression of the protein in the untreated is almost zero. qPCR results show a downregulation (which was expected) of the protein post transfection. qPCR also shows down regulation post treatments with taxol and cpt
Is this result repeatable though? Could it just be a random error somewhere, just saying before you put too much thought into it.
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