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TransMessenger Transfection Reagent Handbook

web site contains siRNA transfection protocols and the most up-to-date information and literature on QIAGEN reagents for RNAi. Visit the Transfection Tools site for answers to your RNAi questions www.qiagen.com/transfectiontools/.

Optimizing siRNA Transfection

To achieve the best results in siRNA transfection we recommend optimizing the following parameters.

Amount of siRNA and Enhancer R
The amount of siRNA used is a critical factor for efficient transfection and gene silencing. The recommended starting amount for transfection of siRNA in 24-well plates is 0.8 g. A pipetting scheme for optimizing the transfection of siRNA in adherent cells in a 24-well format is provided in Table 4 on page 16. Always use a 1:2 ratio of nucleic acid (g) to Enhancer R (l).

Ratio of TransMessenger Transfection Reagent to siRNA
The ratio of TransMessenger Transfection Reagent to siRNA should be optimized for every new cell type and siRNA combination used. As a starting point for optimization we recommend using an siRNA to TransMessenger Transfection Reagent ratio (g:l) of 1:5 when using 24-well plates. To optimize siRNA transfection in 24-well format, prepare separate transfection mixtures using:
The starting point siRNA and TransMessenger Reagent quantities that are given in the protocol on page 16
Half the listed quantities and ratios
Double the listed ratios and quantities.

Cell density at transfection
The optimal confluency for transfection of adherent cells with siRNA is 5080%. The optimal confluency should be determined for every new cell type
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