We would like to transfect post-mitotic neurons cultured from early human fetal neocortex with siRNA. As a trial experiment,we have tried Dharmafect 1 to introduce siRNA against GADPH and found 5% knockdown of mRNA levels at most (measured by qPCR). Has anybody got any tips or recommendations for methods, reagents?
Transfection of siRNA is generally efficient if you have the appropriate reagents and controls. Primary cells usually require more gentle reagents to conserve cell viability (like this for Neuro-2a https://altogen.com/product/neuro-2a-transfection-reagent-neuroblastoma-cells-ccl131/) but overall the small size of most siRNAs makes it easy to transfect in comparison to larger plasmids.
I use Lipofectamine RNAi max from life technologies. It's worked for most of our siRNA studies.
I use Lipofectamine RNAiMax too and i get arround 90% of silencing. You can also try InterferIN from Polyplus Transfecction.
For mouse primary neuron culture, Lipofectamine 2000 worked within 24-48 hours for most of the proteins of my interest. I tried Lipofectamine RNAmax but was not so different or a bit less efficient.
Hi Gavin, I am not sure about what you are trying to achieve but 5% reduction is not bad because you are averaging RNA level of both transfected and non-transfected neurons. An important info that we would need is how much reduction do you see at the IF level, for the transfected cells? If GADH is absent (ie: your siRNA is 100% efficient) then 5% represent the fraction of transfected cells and there is hope that electroporation will increase a bit this fraction but do not count on more than 20-30% with healthy neurons. Good luck
Hi Gavin, you have to think the experiment in two parts, one if the efficience of delivery from the method you decided to used and on the other hand the silencing efficience of your siRNA. How did you assess the efficience of transfection? It could be that the transfection went fine but the siRNA was not efficient enough in the window time you have.
I have tried Lipofectamine2000 and amaxa nuceofector in primary mouse hippocampal and cortical neurons. Liposome-based transfection yields 10-20 % transfection efficience while you can achieve around 40% with nucleofection.
Thanks for all the helpful suggestions. What I'm hearing from you is that Lipofectamine products may be a good bet but that a little bit of electroporation should help (as long as we don't kill our cells). I know we can't expect much more than 30% knockdown but we'd like a little more than 5%. Once we have devised a new protocol, perhaps we will post it for comments.
Also check out our approach based on linking the siRNA to the cell-penetrating peptide Penetratin (see attached paper). We saw good knockdown with less toxicity than Lipofectamine 2000. Contact Carol Troy at Columbia for the latest protocol, the method has been used successfully by many groups at this point.
Article Highly Efficient Small Interfering RNA Delivery to Primary M...
I would suggest using a fluorescent siRNA transfection control, such as BLOCK-IT Alexa Fluor Red. That way you can visualise the efficiency of your transfection against a DNA counterstain (I recommend NucBlue so you can just do it all live and save time). Once you're satisfied with your level of transfection, then you can look at the amount of knockdown you get.
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