Considering a phenotypic assay (proliferation/viability), would you transfect together with the target gene siRNA also negative ctrl siRNAs to achieve the same concentration as the multiple target gene siRNA?
Eg: transfection of siRNA ctrl at 30 nM VS transfection of siRNA-antiA 10 nM + ctrl at 20 nM (total 30 nM) VS transfection of siRNA-antiA (10)+ antiB (10) + antiC (10). Would the three condition be comparable?
Two points are important:
(1) siRNA transfection is temporary (up to few days) and is not integrated into genome (like shRNA)
(2) siControl is composed of random sequences that does not target any gene inside the cells.
As per my knowledge co-transfection of siControl (20 nM) and siRNA targeting a gene (10 nM) vs siRNA targeting a gene alone (30 nM) is OK.
In our lab we have been using three siRNAs (10 nM each) targeting different sequences of the same gene vs siControl (30 nM) alone and that works perfectly.
Moreover in overexpression experiment I have used adenoviral mediated gene transfer where the same type of balance I have maintained.
e.g. Adeno-LacZ (50 moi) + Adeno-TIS21 (50 moi) vs Adeno-TIS21 (100 moi).
FYI: TIS21 is a gene that I wanted to overexpress while LacZ was my control
good luck...
Ciao Chiara,
in my opinion the 3 conditions are indeed comparable: you are using the same concentration of siRNA, what is changing is just the target sequence. Thus, you could infer that the phenotype is the result of selective down-modulation of mRNA target and not the general RNA interference procedure. I guess that siRNA anti-A/anti-B/anti-C are a pool of different siRNAs against the same target sequence.
Ehi, thanks a lot for imput :)
Actually, the three co-transfected siRNAs would be against different genes, not against same target. The aim in the end is to be able to compare the single gene knockdown to the multiple one, without having to plate control at different concentrations.
Regarding the use of negative controls, sometimes they might target genes inside the cell (eg. the ones we are using are known to have effect on EGFR, as it's written in the product page).
Hi Chiara,
You raised an interesting issue of using siRNAs.
Total concentration of siRNA and siRNA/liposome ratio are associated with transfection efficiency. The three siRNA options that you suggested have the same concentration, 30nm. They may have no influence on transfection efficiency. However, siRNA controls not necessarily have no effect on specific target mRNA. They may interfere your observation on effects of specific siRNA. Learning from my experience, setting one or more siRNA controls only (not mixed with specific siRNAs) is required.
Best wishes,
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