Small interfering siRNA (siRNA) is a class of 20-25 nucleotide double-stranded RNA molecules, which interfere with the expression of specific genes with complementary sequences, leading to RNA interference (RNAi). Synthetic siRNAs may be used for knocking-down the expression of specific genes.
The success of an RNAi experiment mainly depends on three variables: 1) the transfection efficiency 2) the silencing efficiency 3) non target (unspecific) effects. Therefore, the use of the appropriate positive and negative controls is fundamental in planning and executing and RNAi experiment, as well as in analyzing and interpreting the results.
Since the silencing efficiency of Silencer® GAPDH siRNA (human, mouse, rat) has already been tested, the success of the RNAi experiment depends on the transfection efficiency only. Therefore, it may be used for developing and optimizing the siRNA transfection conditions (cell number, concentration of siRNA, amount of transfection reagent). To determine the best transfection conditions in a primary culture of rat aortic endothelial cells, I transfected them with different combinations of Silencer® GAPDH siRNA/tranfection reagent, and I evaluated the expression of Gapdh 24, 48 and 72-hours after transfection by real time RT-PCR. The Gapdh mRNA levels in each sample were compared to the levels measured in cells treated with the transfection reagent only (mock control) and normalized vs. 18S rRNA. I chose the combination of siRNA/ transfection reagent giving the lowest toxicity and the highest down-regulation of Gapdh mRNA. In my hands, the silencing efficiency was very high (more than 70%) in any sample analyzed.
I also use the Ambion Silencer® GAPDH siRNA (human, mouse, rat) in any RNAi experiment to control the transfection efficiency (positive control). Moreover, to control for non-target (unspecific) effects, I transfect the cells with a commercially available non-silencing siRNA sequence, which has no significant homology to any known mRNA sequence for mouse, rat, or human (negative control). The Silencer® siRNA (human, mouse, rat) and the negative control may be also purchased together in a convenient “starting” kit (Cat.#AM4624).
The main advantages of using Ambion Silencer GAPDH siRNA (human, mouse, rat) are: 1) the siRNA is highly efficient, 2) the kit is highly versatile: the siRNA sequence may be used in rat, mouse and human cells, 3) it is ready to use and 4) it’s not too expensive. The main disadvantage is that the silencing sequence is not known.
In conclusion, I would recommend using Ambion Silencer GAPDH siRNA (human, mouse and rat) as positive control in siRNA experiments, particularly to those who routinely work with cells obtained from different species.