I tried a pool of 3 siRNA transfection in Hela cells using RNAiMAX but all of of them failed. 100nM conc siRNA and opti-mem were used at 40~50% cell confluency and then 6 hour later, added DMEM (20% FBS) and harvested cells after 48 hours of transfection.
(I checked tranfection efficacy with qRT-PCR.)
First of all, siRNA reduce protein expression via translation inhibition (NOT mRNA transcription). Secondly, via using qRT-PCR you may check inhibitory but not transfection efficacy. I would propose to (really) check transfection efficacy (e.g. via transfection of dye-bearing short RNAs into cells and ameasure number of molecules (or signal due to the dye) in cells, e.g. via FACS (for ref. see e.g. J Hypertens. 2016 Feb;34(2):323-31. doi: 10.1097/HJH.0000000000000799).
This could help you to assess whether your method/protocol works.
In my opinion, the described problems could be related rather to the protocol or the experimentator, than to the siRNAs.
Possibly, siRNAs were insufficient to reduce expression of the target molecule. However, this should be tested on protein level (e.g. via WB). Alternatively, the potential binding of siRNAs to the mRNA target sequence should be checked in silico.
Hi! In my experience, HeLa cells are very easy to transfect with siRNA. We were rutinely using Oligofectamine, but RNAiMAX should work too (maybe even better). I'm using siRNA at 30 nM - 50 nM final concentration, but that should not affect the result. As Andreas proposed, I would try to estimate the knockdown efficiency by western blot. Also, why do you use 20% FBS? Hela normally grow in 5-10% FBS, 4.5 g/L glucose. I don't know if the FBS concentration can affect the transfection efficiency though. Some proteins are long lived so you can as well try to transfect your cells at 30% confluency (viable with Oligofectamine) and culture them for 72 hours before harvesting. Good luck!
Hi Hansong!
For standardizing your siRNA transfection you should start with a range of siRNA concentrations say from 20-50 nM rather than a single concentration.Hela cells are ideal for transfection and the cells should be healthy and properly viable to uptake the siRNA. Follow the transfection protocol of the manufacturer and maintain the incubation periods thoroughly. Lastly, the best method to check efficiency of gene silencing is by western blot. I have performed transfection experiments with siRNA and found these are the pressure points.
I agree with the aforementioned comments regarding testing of optimal conditions (e.g. concentrationof siRNA or the transfection reagent etc.).
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