Hello!
I've been trying siRNA knockdown using RNAiMAX (invtrogen) but I'm not getting any significant knockdown in the T47D and HCC-1954. I'm using forward transfection.
The protocol I uses is:
1. seed 150,000 cells/ well in a 6 well plate. Allow to attach for 24hrs.
2. Replace media with antibiotic free media the day after and add the lipid-siRNA complex.
3. I'm using 10nM siRNA concentration and 9uL RNAiMAX/ well. I'm not having much luck.
I've used the same protocol for MDA-MB-231 and it works.
Would anyone have any suggestions? In particular regarding the volume of the lipid. I'd prefer to keep the siRNA conc. to 10nM as we're low on siRNA .
Thank you in advance for all your help.
Hi,
You can use 1mL media/well during transfection on a 6-well plate (250uL lipid-siRNA complex + 750uL media). This will increase the siRNA concentration to 25nM. After 4 hours of transfection (which is usually sufficient for peak siRNA uptake), you can replace or add more media until you measure the knockdown (ideally 36 - 48 hours after transfection).
25nM siRNA Treatment:
3uL siRNA (10uM) + 150uL OptiMEM = 150uL siRNA (200nM)
+ 9uL Lipofectamine + 150uL OptiMEM = 300uL siRNA (100nM)
250uL siRNA (100nM) + 750uL Media = 1000uL siRNA (25nM)
Cost-effective siRNA:
If you know the sequence of siRNA, then you can order siRNA through Sigma's Custom siRNA Oligos service. By ordering larger quantity you can save a lot, i.e. 50 OD = 250nmol = 25mL of 10uM siRNA.
50 OD, HPLC purified (In vivo Grade), lyophilized duplex siRNA, with or without fluorescent labels, with [dT][dT] overhangs would cost less than 1000 EUR (price could vary depending on the country). It would be worth an investment given the amount of siRNA would last for years. Best thing is just to ask for a quotation, even if you are not able to purchase immediately.
https://www.sigmaaldrich.com/life-science/custom-oligos/sirna-oligos.html#siRNA
Best Wishes.
That’s great. Thanks so much Subaash. I’ll try your suggestions.
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