I am starting a experiment where i wish to use siRNA to target a gene in MCF-7 cells.
I have a pre-validated siRNA and a negative scrambled control from Ambion.
I began my optimization by using a range of siRNA:transfection reagent ratios and anaylzed the gene expression using semi-quantitative RT-PCR. However, i have now noticed that alot of people seem to use real-time and i was wondering if my optimization results will be accurate enough to allow me to proceed with the next stages of the experiment or should i do a real-time pcr to confirm?
Also i was just wondering if its necessary to have a positive control as i don't want to do all the work & then be told that i should have had one!
Many thanks in advance!