I am planing to perform a follow up screen to RNAseq most likely in adult DRG neurons or hIPSC derived neurons. I am looking at the flexiplate siRNAs from Qiagen (100pmol/23USD) or Mission Pre-Designed esiRNA from Sigma (75pmol/17USD).
Any experience with either?
Other possibilities?
I have had good results with the Sigma ones in easily transected cancer cells. I am however wondering whether you have considered any retroviral shRNA libraries so you can knockdown using viral transfections (if practically possible of course)? Especially neurons can be tricky to transfect efficiently so you will increase your changes of getting a successful screen.
I would echo Patricia's answer. siRNA transfection in primary neurons is very difficult and typically does not yield acceptable levels of knockdown. Retroviral or lentiviral shRNA approaches are much more feasible.
Our lab has switched from transfection to electroporation of primary mouse cortical and hippocampal neurons, and some have shown >50% of cells express GFP with this method (see Dityateva G et al J Neurosci Methods. 2003 Nov 30;130(1):65-73. for one report). We have not used this technique for siRNA experiments, but the neurons are more tolerant to electroporation than Lipifectamine based transfection.
I can second the suggestion that you consider a viral system. It's a bit of work at the outset, but typically results in 80-90% infected cellls in cortical primary neurons.
Thanks a lot, I am aware of the benefits for the viral infection but I am limited to this approach because of money and time reasons. Also I want to screen several hundred constructs and constructing an shRNA library is too time consuming. Or is it possible to acquire a budget shRNA library?
Yes, in general shRNA products will be more expensive than siRNAs if you are purchasing either glycerol stocks, plasmid, or transduction particles. If you are constructing an extensive screening library, you can often negotiate for a lower price.
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