I am a graduate student partly done with my second year and I am currently writing my comprehensive exam proposal. In my proposal, I need to talk about some experiments where I do siRNA mediated knockdown of certain genes. I am new to this field and I am wondering what are good methods to validate that I am knocking down the right gene, and not simply observing offtarget effects. I've read about doing rescue experiments by transfecting in siRNA along with constructs that are mutated as not to be recognized by siRNA. Is this the gold standard for validation that siRNA is definitely targeting mRNA of interest?
I've also read about using 3'UTR targeted siRNA so that rescue experiment could be done with wildtype contructs. I am rather unclear about this point. I thought siRNA is normally targeted to 5' end and if it targets 3' UTR, is this still effective? Is this method commonly used?
What other experiments could be done to validate siRNA is targeting the right mRNA?
If siRNA doesn't work, should shRNA be used to achieve stable knockdown?
I am very new to this field, so please correct me if I am saying things are are completely wrong. Any help would be greatly appreciated.