experimental design - (Feb/22/2012 )
I have a question that didn't seem to fit in the categories listed in the bioforum so I'm posting it here under general lab techniques.
I have created some stable trangenic cell clones that over express gene X. I understand that ideally any changes observed in these cells ought to be confirmed in a second cell clone of that type to ensure we're not seeing clone-specific effects....
However, from screening the set of clones for whether they've got the overexpression, there was only one that showed heightened expression. The others were around 1 fold less than the desired minimium level of expression or had hardly any expression. My question is, is there any articles (or suggestions anyone may have) for how I can get around the '2 clone minimum' validation to be sure that any changes I see in this single transfected cell line are not due to a clone specific effect? It's taken months to reach this stage and I don't particularly want to start from scratch at this point with a new transfection expt.
Problem is now solved after speaking to another PhD student. Thanks.
Please share =)
Essentially, the qPCR screen I did of the transgenics clones did in fact reveal some that were within 0.5 fold of the normaliser sample I had. qPCR will not give 100 % accurate quantitation of the expression level of the gene of interest anyway, so when I thought there was only one that over-expressed compared to the normaliser there were in fact three (2 clones exhibited expression in the range of the normaliser when looking at the 95% CI's). I hope this explains it. :-)