Protocols
Mammalian Protocols
A critical parameter for a successful high throughput RNAi screen is optimization of transfection conditions. Because siRNAs and dsRNAs are prealiquotted into plates, screeners must optimize reverse transfection conditions, where transfection complexes are added to the cells while they are in suspension. Some conditions to consider are cell number, type and amount of transfection of reagents, and siRNA or DNA concentrations. An example of an optimization experiment is listed below.
Also we have found that transfection conditions for siRNA with DNA are different from transfecting siRNA alone. As a result, specific optimization will be required for co-transfection versus siRNA transfection.
While labelled siRNAs can provide one with a qualitative sense of transfection efficiency, ultimately, the use of positive and negative assays controls is best for optimization. Advice on siRNA transfection can also be found on various vendors websites (e.g. Ambion, Dharmacon, and Qiagen).
Protocol for 384-well reverse transfection of 293T cells with siRNA and DNA using siPORT NeoFX (Ambion)
Add 0.15ul NeoFX to x ul of OPTI-MEM.
Incubate 10 minutes.
Add x ng of reporter DNAs.
Mix, add 5 ul of diluted NeoFX and DNA to the well containing 5ul of 0.3uM siRNA pool.
Spin down plate; incubate 10 minutes.
Add 40ul of cells (1500 cells/well).
Spin down plate.
