Hi fellow researchers. Accell siRNA is claimed (by the company) to be good for use with primary cells (hard to transfect type of cells). The concentration of siRNA recommended for use is ridiculously high, 1µM, without the use of any transfection agent, just a delivery medium provided by the company. So far knockdown efficiency (mRNA level) of cyclophillin B (positive control) seems ok unlike the target gene, against ciliogenesis. Has any one tried to use a transfection reagent on primary cells? When do you think the best time to transfect in order to stop cilia formation (during proliferation or differentiation)?
Our lab has consistently achieved excellent knockdown using Permute (Signa Labs)... http://signagen.com/In-Vitro-siRNA-Transfection-Reagents/SL100566/PepMute-siRNA-Transfection-Reagent
We use primary mesenchymal stem cells and have knocked down over 10 targets successfully with it. Here are some manuscripts with published siRNA results using this reagent...
http://www.ncbi.nlm.nih.gov/pubmed/23836527
http://www.ncbi.nlm.nih.gov/pubmed/25787126
http://www.ncbi.nlm.nih.gov/pubmed/23821483
As far as the best time to transfect to stop primary cilia formation, I would think that proliferation should be the target time, as the cytoskeletal structures form early, and aren't necessarily a result of differentiation (which of course depends on the cell type and the differentiation status).
Thank you William ... I checked their website I will try to order free testing samples and will see what happens. I use primary renal cells, isolated from the kidney and cultured for 8 days max before wearing off. Is it an idea to transfect them right after isolation or later during culturing period?
Accell works well in many cell lines. Couple problems (i) off-target effects due to high concentration, in some cases (ii) pretty high cost
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