Ready-to-use Optimized Protocols for siRNA Nucleofection

For the transfection of siRNA oligonucleotides into your specific cell-type, we recommend using the respective cell-type specific Nucleofector Kit (standard or 96-well ) and follow the Optimized Protocol (for more information on the available Nucleofector Kits please see our product section or our Cell Database).

Optimal nucleofection conditions for a particular cell type are identical whether you are transfecting DNA or RNA. So we recommend the following steps:

First, perform a preliminary experiment with pmaxGFP (our positive control plasmid, included in every kit) in order to establish/verify the optimal nucleofection conditions for your cells.
Next, use the identical conditions for your siRNA experiments, but replace pmaxGFP with your siRNA oligo. You may wish to include a sample with pmaxGFP in your siRNA experiments in order to measure the success of nucleofection or you can even co-transfect pmaxGFP and your siRNA within the same sample. However, if you are using this as a means of estimating transfection efficiency for your siRNA, do keep in mind that the transfection efficiency for siRNA duplexes is even higher than for plasmid DNA.

Positive controls for easy establishment of siRNA nucleofection:

Co-transfection of pmaxGFP with an siRNA against maxGFP mRNA ( siRNA Test Kit – For Cell Lines and Adherent Primary Cells)
Down-regulation of a house-keeping gene (e.g. Optimized Protocol for down-regulation of endogenous genes such as CD2, CD4 and vimentin in primary blood cells)
Experiments with fluorescently labeled siRNAs have shown transfection efficiencies of up to 99%. Generally, unless a confocal microscope or FACS is available, the use of fluorescently labeled siRNA for initial set-up experiments is not advisable as many fluorescent labels fade quickly and microscopic analysis may lead to false positive results.

Stability of siRNA duplexes in the Nucleofector Solutions: The standard and 96-well Nucleofector Solutions were tested for RNAse activity. Incubation of RNA in the solutions for 2 hours at 37°C did not affect RNA stability. High siRNA transfection efficiencies can only be achieved with the amaxa certified cuvettes, included in the cell-type specific kits.