I have to deliver two different siRNA at the same time to cancerous cell culture whilst satisfying efficiency.
Do you have a shRNA encoding plasmid similar or equal to one of your siRNAs?
If yes, you can transfect first the plasmid (maybe trying a stable transfection) and transfect the other siRNA afterwards. For my knockweledge the shRNA encoding plasmid has a longer half-life, so you can collect your cells at an appropriate time point (e.g.: shRNA transfection at day 0 , siRNA transfection at day 3 and cell collection at day 5)
If not, I tried only once a double-siRNA transfection with HiPerFect from Qiagen (but I think you tried a similiar way , too):
I took 15 nM (for 2*10^5 cells per well in a 6-well) each of my siRNAs, dilute in 100 µl FCS- and antibiotic-free medium, short vortex and add 50 µl HiPerFect, incubate for 15 min and pipette the solution on my cells. My cells were at this time point in FCS- and antibiotic-free medium. I Incubate over night, add normal growth-medium and incubate for 48 h. I collect my cells after 72 h.
What did you try so far?
Kind regards
Hi Aref,
First you could find the optimal concentration of each siRNA to have highest efficiency. Then try to add both siRNA in same time. if this method doesn't work, i recommend you to give a try with " reverse transfection" method. In this method, first you must prepare transfection reagent, and then add cell suspension in it. (cells will not be seeded in plate for overnight or some hours in advance.)
Good Luck.
Do you have a shRNA encoding plasmid similar or equal to one of your siRNAs?
If yes, you can transfect first the plasmid (maybe trying a stable transfection) and transfect the other siRNA afterwards. For my knockweledge the shRNA encoding plasmid has a longer half-life, so you can collect your cells at an appropriate time point (e.g.: shRNA transfection at day 0 , siRNA transfection at day 3 and cell collection at day 5)
If not, I tried only once a double-siRNA transfection with HiPerFect from Qiagen (but I think you tried a similiar way , too):
I took 15 nM (for 2*10^5 cells per well in a 6-well) each of my siRNAs, dilute in 100 µl FCS- and antibiotic-free medium, short vortex and add 50 µl HiPerFect, incubate for 15 min and pipette the solution on my cells. My cells were at this time point in FCS- and antibiotic-free medium. I Incubate over night, add normal growth-medium and incubate for 48 h. I collect my cells after 72 h.
What did you try so far?
Kind regards
Mixing is not of itself a problem . Dharmacon actually sell mixtures of siRNA "SMARTpools". But you will need to optimise concentration
I have often used double transfections… two different siRNAs targeting two different genes and this worked really nicely. I have used this as a strategy to do rescue experiments (my protein of interest inhibited another protein so by knockdown of both proteins I restored the original effect and could see efficient knockdown of both). Using shRNA I had less luck so I guess this might block the dicing machinery to process shRNA using excessive amounts of such constructs. To do double knockdown experiments I first optimise the protocol … figure out when you see maximal knockdown with the lowest amount of siRNA transfected and combine the lowest amounts for each siRNA in the final experiment. I do not increase the amount of hiperfect (or lipofectamine) or the amount of optimem. I have also used double siRNAs in nucleofections and that works fine as well.
My protocol for hiperfect in 6 well dishes would be as follows:
Combine 0.25-2.5 ul of 20uM of each siRNA (first test knockdown individually using the same protocol) in 800 ul of opti-mem (serum free). Add 7.5 ul hiperfect and vortex. Leave for 15-20 minutes at RT. Add this mixture drop wise to 1.5 ml of refreshed medium on your cells. I have done this both reversed and forward dependent on the cell line… e.g. while splitting the cells and adding the transfection reagent before the cells attach, but most frequently I have used this after the cells have settled for 1 day and are at a density of about 40-60 %. Dependent on the turnover of the protein that you are interested in you should look after 1-3 days for the effects to take place. Off course this can be a bit tricky if you are looking at two genes with massively different turnover times.
For nucleofections I have used the AMAXA protocols in which each cell line has its own optimised protocol. Most of the optimisation has already been done by Lonza or other researchers and can be found here: http://bio.lonza.com/6.html
The advantage of the nucleofection is that you can also do a simultaneous over expression easily. If you do a knockdown of your gene of interest you can easily transfect your gene of interest to rescue the phenotype. I hope this helps, but feel free to ask if anything is unclear.
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