Easy/difficult to transfect?
Any recommendations for companies and products?
Thank you in advance!
I've never tried to transfect that cell line BUT I've never had a problem to transfect siRNAs. DNA is a totally different story but siRNA transfection works all the time. Go ahead and try to transfect it with either Lipofectamine RNAiMax or Lipofectamine2000, it should work. I've used those 2 transfection reagents to transfect a wide range of cells.
If you have problems transfecting, I suggest you can get a flourescent siRNA and try Calcium Phosphate or electroporation to deliver the siRNAs, and then use a flowcytometer to check if your cells introduced the fluorescent siRNA.
I hope this helps :)
Excellent :D that does help a lot, much appreciated!Will try them. For a control would you recommend scramble siRNA or one of those kits that you can buy from e.g. dharmacon or invitrogen?
Thanks,
Sophie
Thanks Joanna :) I will try the RNAimax thanks so much for the info :)
Sophie
No problem,
The best siRNA control is one that do not target any other gene. Almost every company has one non-targeting siRNA negative control. Just make sure it is the same kind of siRNA as the one used to knock-down your Gene of Interest. If you use a Dicer Substrate siRNA (Like the ones from IDT-DNA) to knockdown your gene then use Dicer Substrate negative control too. The same goes for other siRNAs. Make sure to use non-targeting siRNAs of the same length and same modifications as the one used for your Gene of Interest.
I hope this helps. Happy knocking-down!
Hello Sophia,
In general it is always depending if they are easy or difficult to transfect. Many companies offer optimized transfection reagents for primary or hard to transfect cells.
In general you have to use a reporter plasmid (GFP) and try out what transfection reagents you have available in your lab.
If you have to order there are many vendors out there selling suitable transfection solutions. I used RNAiMAX but I can not say if it will work well with your aortic cells. For testing which reagent will work you can enquiry for test vials on the homepage of many suppliers like:
https://www.thermofisher.com/at/en/home/brands/product-brand/lipofectamine.html
http://www.lonza.com/products-services/bio-research/transfection/nucleofector-kits-for-primary-cells.aspx
https://www.clontech.com/
https://at.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/transfection/
In a practical course we worked with endothelial cells and used calcium-phosphate transfection with a plasmid which worked very efficient but I do not know if it works that good with small siRNAs.
I hope this suggests help you.
Regards
RNAi Max from Invitrogen (now thermo scientific).......use their recommended conditions.........it works awesome for siRNAs in any type of cell line.
Best of Luck..
Thanks Volker lots of helpful advice :)
Thank you everyone for the replies.
Will give it a go and write what works for me once figured it out!
Best wishes,
Sophie
For this can of hard to transfect cells I will recommend Magnetofection especially SlienceMag specific for siRNA
you can see publications and results here (click on citation database):
http://www.ozbiosciences.com/transfection-sirna/65-silencemag-transfection-reagent-cell-silencing.html
If I can further assist you, let me know at [email protected]
Good Luck
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