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 an RNAi experiment, as well as in analyzing and interpreting the results.
I apply the RNAi technique to study the role of specific transcription factors in the induction of protective genes in human skin fibroblasts or in rat endothelial cells. To control for transfection efficiency and silencing efficiency (positive control), I normally use a thoroughly tested siRNA sequence directed against a highly expressed gene (e.g. GAPDH). Instead, to control for non-target (unspecific) effects, I transfect the cells with a non-silencing siRNA sequence, which has no significant homology to any known mRNA sequence for mammalian genes (negative control). If altered expression or phenotype is observed in cells transfected with negative control siRNA, these are not due to sequence complementarity. Since the main read-out of my experiments is the modification of the expression of genes, which are controlled by the transcription factors under study, I need a thoroughly tested and validated negative control to correctly evaluate the results.
I used AllStars Negative Control siRNA from Qiagen for determining unspecific effects of siRNA tranfection in my experiments. Briefly, I transfected the cells with a siRNA targeting a transcription factor, with a siRNA targeting GAPDH mRNA (positive control), with AllStars Negative Control siRNA from Qiagen, or with tranfection reagent only (mock control). Cells receiving just the transfection medium without siRNAs and transfection reagent were also included in the experimental setup (untreated control). After 24, 48 and 72 hours, the mRNA levels of the targeted transcription factor were evaluated by real-time RT-PCR and compared to the levels measured in the mock control (to evaluate the effect of the transfection reagent), as well as to the negative control (to evaluate the non-targeting effects). In my hands, the expression of the targeted genes was similar in both mock control and cells transfected with negative control siRNA.
The main advantages of using AllStars Negative Control siRNA from Qiagen are: 1) the siRNA is thoroughly tested and validated for non specific effects on gene expression by microarray analysis, 2) the kit is highly versatile: the siRNA sequence may be used in different species, 3) it is ready to use and not too expensive. The main disadvantage is that the silencing sequence is not known.
In conclusion, I would recommend using AllStars Negative Control siRNA from Qiagen as negative control for non-targeting (unspecific) effects in any RNAi experiment, particularly if the main read-out of the experiment is the modification of gene expression.