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RNAi: The Controls You Need

hp?"> Validated siRNAs were tested for their ability to knockdown target mRNA levels. All of the siRNAs tested were effective (>70% expression reduction) at 10 nM, and five of the seven were effective at 1 nM.

Figure 1. Titration of Silencer Validated siRNAs. The indicated siRNAs were complexed with siPORT Lipid Transfection Reagent (Ambion) and the resulting complexes were added to HeLa cells in 24 well plates at the final concentration of siRNA shown. 48 hrs after transfection, RNA from the treated cells was recovered and reverse transcribed using the RETROscript Kit. Target cDNA levels were measured by real-time PCR. Expression of target genes in the transfected cells was compared to cells transfected with an equal concentration of Silencer Negative Control #1 (Ambion). Input cDNA in the different samples was normalized using real-time data for 18S rRNA. The bar graphs represent an average of three data points.


Validation Via Array Analysis and Rescue Experiments

To better understand an siRNA's effect, global gene expression patterns can be analyzed (e.g. using microarray analysis). Differential expression patterns can be compared between non-transfected cells, cells transfected with a negative control siRNA, and cells transfected independently with 2 or more gene specific siRNAs. Yes, it is time consuming and can be expensive, but this ultimate control gives the best indication of specific versus non-specific siRNA effects.

Rescue experiments in which the
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5. Designing Controls for siRNA Experiments
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