So I have been doing some experiments to try and characterise the function of an uncharacterised gene. I have knocked it down using an siRNA I bought from Life Technologies (>90% knockdown validated by both qPCR and Western Blot), and found that knocking it down appears to inhibit the inflammatory pathway. When I subsequently stimulated with TNF or IL-1, the transfected cells have lower expression of inflammatory markers than TNF stimulated untransfected or scrambled control transfected cells.
the problems came about when I tried to validate this using a further 2 siRNAs also purchased from Life Technologies to the same gene. Transfection with these siRNAs knocks down the gene of interest by similar amounts, but doesn't produce the same effects on the inflammatory markers when the cells are stimulated.
The most obvious explanation is that these results are due to some off-target effects by the original siRNA disrupting another gene involved in inflammatory signalling somewhere (which throws a few months of research into some interesting results out the window), but I would like to know if it is possible to identify the other gene(s) that the siRNA is affecting. When I BLAST the siRNA sequence the top result is my gene of of interest with all bases in common, then one gene with 17 bases in common, and after that several with 15 bases. I get a similar pattern with my other two siRNAs, but with different genes with 17 or 15 bases in common.
Is this a reasonable way to try and identify the other gene(s) that is being knocked down? Or would it be simpler just to send some samples off for RNAseq and wait for the results. And also if there are any recommended publications to learn more about this.
I am mainly asking as most of the literature seems to deal with large scale siRNA screens involving 1000s of self-designed siRNAs, rather than severe off target effects from commercial sequences in much smaller scale experiments.
Edited by philman, 16 September 2015 - 08:22 AM.