Hello,
I am still new to doing RNAi experiments,
after I transfected siRNAs against my gene of interest, using siGAPDH as a positive control as well as a commercial negative control siRNA as a negative control, do I change the media after 24 hours?
I noticed quite a lot of cells death across all treatments - is this normal? I plan for this experiment to go on for a total of 110 hours measuring cell number every 24 hours. I expect that although a lot of cells died, the genes I'm interested in will have significantly less cells than the negative control over the five days. I am collecting RNA for RT-qPCR to confirm knockdown as well everytime I count the cells.
What do you do usually, and how can I further improve this sort of experiment?
Thank you!
During transfection, don’t add antibiotics and it causes cell death. You can do, one cell culture well without treatment anything(just put media) and another well use RNAiMAX reagent. Then you can easily measure percentage of death cells for RNAiMAX lipofectamin reagents. And another thing, in my experience, reverse transfection is determental for some cell lines but its work more efficient compare to the forward transfection.
Best of Luck, Stanley
have you optimized the transfection yourself to obtain efficient transfection with minimal toxicity? If not, I'd start there with siGAPDH and/or fluorescently labeled siRNAs before moving to experiments targeting your gene of interest.
What kind of cells are you using? I wouldn't incubate siRNA / RNAiMAX with cells for longer than 24 hours, it is known to be quite toxic. Have you optimised the doses of transfection reagent and siRNA? You'll need to make sure that there is minimal toxicity or the cell death would obscure the magnitude of gene knockdown. I would recommend optimising first with cytotox assays. Your option might have to be multiple doses of siRNA over the 110 hours if it proves too toxic but you will have to validate the time-course of knockdown.
Hope this helps.
Thank you all for your replies, it seems the consensus is that to optimize toxicity first. I will try that, thank you!
Hello! no matter which reagent you use for your siRNA transfections, you will always need optimization. As an example, you can have a look to the manual for our Viromers BLUE and GREEN (attached file), it drives you through optimization, pages 9-13: playing on transfection complex amounts, reagent:siRNA ratios, cell density, dose-response curve.
Depending on your cells, specific protocols could have been reported by other people, please tell us which cells you use.
By the way, if you want to try our reagents, do not hesitate to ask me, I can provide test samples for free.
Bests, Sandra
Hi
I also tried Lipofectamine® RNAiMAX Reagent with epithelial cells and observed a cell death of 5-10%. The Invitrogen (Waltham, MA, USA) protocol doesn’t advise to change the media. Has anybody tried the 25pmol of siRNA in different culture media volumes for a well of the 6-well plate (i.e. 1mL, 2mL or 2.5mL)? As the protocol doesn’t exactly advises to maintain 25pmol of siRNA in exact volume, I feel increasing the media volume may reduce the cell death after the transfection.
Any suggestions ?
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