Hello,
I was wondering if someone could help with siRNA transfection. I'm trying to lipid transfect HEK 293T cells using Qiagen's hiperfect transfection reagent. I have been working from the protocol on the Qiagen website; 20nm siRNA/control, 12μl hiperfect and 5X10^5 cells per well in a 6 well plate. I have repeated this numerous times, triple checking calculations, techniques and reagents, all to no avail. I then spoke with a colleague of my supervisor who worked with this technique previously, he suggested doing a double transfection (transfect day 1, day 2, change medium day 3, count cells on days 4+). I tried this and still no results. The cells were looking highly confluent after 2 days so this person suggested using 1.5X10^5 cells per well as this gave him better results. I have been getting silencing in every single well (siRNA, mock transfection, + and - controls) except the untransfected wells, suggesting the transfection reagent may be having some toxic effect on the cells. I repeated with 8μl transfection reagent and there is now a slight downregulation in the amount of + control cells (apoptosis genes induced) which is what I would expect.
I am currently repeating this experiment ft 1X10^5 cells per well with 4μl transfection reagent and if this doesn't work I think I will move on and try with the shRNA silencing of my gene of interest.
I was wondering if anyone has tried silencing this line before and had similar problems? If anyone can offer any advice I would be very grateful!
Thank you.
Should be pretty easy to transfect siRNA into HEK293, given that its one of the most easily-transfectable cell lines. That being said, following protocol is crucial, and having appropriate lipofection reagents (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/) can help ensure the 80%+ results that are standard for HEK293.
HEK 293 cells are very hard (like, very very very hard) to transfect with siRNA or any kind of RNA-based molecule alone (without DNA to carry it). Up to date, the only transfection reagent that shows an average/good transfection efficiency is RNAiMax from Invitrogen. I can only suggest to try shRNA. I have put a similar answered question in researchgate for reference. Another approach, if even shRNA do not work, would be to cotransfect your siRNA with an empty plasmid DNA (empty pSG5 for example), just for the sake of adding a carrier with your siRNA, as a co-transfection of DNA and siRNA seems to work better.
https://www.researchgate.net/post/How_do_you_co-transfect_DNA_and_siRNA_into_HEK_cells
HEK-293 is pretty easy to transfect with, just follow the protocol is fine. Pooling is helpful on reducing off-target effect, and make sure to have the non-specific RNAi the same time. If you have positive control, will be even better.
Dear Sarah,
I can recommend another transfection reagent called Lullaby it has been successfully used in many cells including HEK. You can see the citation here: http://www.ozbiosciences.com/transfection-sirna/24-lullaby-sirna-transfection-reagent.html
and here is one example: http://www.ncbi.nlm.nih.gov/pubmed/24198819
Moreover, the CRUK has selected this reagent for their screening strategy. Here is the information: "We initially collated a transfection reagent library of 26 reagents...By far, our preferred reagent is Lullaby from OZ Biosciences. We have used this reagent in over 20 cell lines and have found it essential in enabling siRNA screens in hard to transfect cell lines..., with minimal toxicity" from the publication: http://www.ncbi.nlm.nih.gov/pubmed/24661213
Let me know if you want to try it, I can send you a sample of our transfection reagents at [email protected]
Good luck
Olivier
There is also DharmaFECT transfection reagent you might want to try for Heks. And yes, with HEKs double transfections are difficult because they are confluent by the time you do the second one. In my experience plating at low density doesn't really work for this cell line for several reasons.
Thank you very much everyone, these answers have all been very useful in allowing me to ascertain what has been going wrong with my transfection, thank you.
Should be pretty easy to transfect siRNA into HEK293, given that its one of the most easily-transfectable cell lines. That being said, following protocol is crucial, and having appropriate lipofection reagents (https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/) can help ensure the 80%+ results that are standard for HEK293.
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