This is certainly true for RNA interference experiments using siRNAs. For this article, we asked Applied Biosystems Research and Development personnel for advice on the proper use of controls for siRNA experiments. Here are their recommendations.
1. Always include a positive control siRNA to monitor transfection efficiency.
Good transfection is absolutely essential for effective target knockdown using siRNA, thus, it is important to include a positive control siRNA in each experiment. The positive control siRNA should elicit reproducible, easily measured knockdown in the cells used in your study. If you see maximal knockdown with this control, you know that knockdown measurements from other siRNAs tested on the same day are reliable.
2. Understand how much knockdown you should see from your positive control siRNA under optimal transfection conditions.
The data shown in Figure 1 illustrate the utility of including a positive control siRNA in your experiments, as well as the importance of understanding what level of knockdown represents “good” transfection. This experiment included an siRNA for GAPDH that routinely elicits ≥97% target knockdown when it is effectively delivered into HeLa cells (≤3% remaining gene expression). The data in Figure 1 show knockdown from an identical experiment repeated on two different days. In one instance, represented by grey bars, transfection was suboptimal: ~8% remaining gene expression was seen from the GAPDH positive control siRNA (92% knockdown). Note the poor performance of other siRNAs in the experiment when the GAPDH control siRNA yielded only 92% knockdown. Had we not known to expect ≥97% knockdown from this control, we might have erroneously concluded that transfection worked fine in this experiment, yet only 24 of the 39 siRNAs tested gave ≥70% knockdown—a rather poor success rate! Note in the second experiment with the exact same siRNAs, the GAPDH control siRNA induced >97% knockdown (Figure 1, green bars). Likewise, all but one of the other siRNAs tested knocked down their targets by 70% or better.
Figure 1. Knockdown Data Showing the Importance of Using a Positive Control to Monitor Transfection Efficiency.
Remaining gene expression after transfection with the indicated Ambion® Silencer® Select siRNA was measured using TaqMan® real-time PCR. The results were normalized to expression in cells transfected with a nontargeting negative control siRNA. The experiment was conducted twice, as shown with grey bars and green bars. Note that in the experiment where knockdown with the GAPDH siRNA transfection control was poor, knockdown with the remaining siRNAs was below the threshold considered to be useful, ~20–30%. In the other instance (represented with green bars), knockdown of GAPDH was in the expected range (>97%), indicating that transfection occurred a high efficiency, and knockdown from the remaining siRNAs was also sufficient for meaningful results.
3. Use unlabeled siRNA as a positive control for transfection.
In theory, siRNA labeled with fluorescent dye should make an ideal transfection control for siRNA experiments: just transfect, and then determine the percentage of cells that have internalized the fluorescent dye. In practice however, considerable variability is seen in the correlation between observable uptake of fluorescently-labeled siRNA and knockdown of the corresponding target. Because of this observed variability; we recommend using unlabeled siRNA as a positive control for transfection.
4. Evaluate knockdown of the positive control target at the RNA level.
There are simple, sensitive protein assays for many positive control targets, and the ultimate goal of siRNA knockdown experiments is to reduce the target protein quantity. Therefore, researchers sometimes question whether it is better to evaluate the positive control transfection by looking for a reduction in the amount of target mRNA or the corresponding protein product.
Because siRNA exerts its effects at the mRNA level, it is more direct to measure knockdown of the target mRNA. We recommend evaluating mRNA knockdown using TaqMan® Gene Expression Assays
in reactions powered with TaqMan Gene Expression Master Mix for robust, reliable results. For experiments using cultured cells, the TaqMan Gene Expression Cells-to-CT™ Kit
(which includes master mix) enables real-time RT-PCR directly in cell lysates without first isolating RNA.
5. Include nontargeting siRNA and nontransfected cell negative controls.
In RNAi experiments, negative controls are just as important as positive controls for obtaining meaningful data. Always include a set of transfections with an equimolar amount of at least one nontargeting negative control siRNA—data from these crucial transfections serve as a baseline for evaluation of experimental target knockdown. For example, the data in Figure 1 have been normalized to expression of the indicated target in cells that were transfected with a nontargeting negative control siRNA, in this case, Ambion® Silencer Select Negative Control #1 siRNA
Nontransfected or cells-only negative controls are also very useful in siRNA experiments. By comparing expression of a housekeeping gene among cultures that were not transfected and cultures transfected with a nontargeting negative control siRNA, valuable information about the effects of transfection on cell viability can be obtained.
Rajeev Varma, Susan Magdaleno • Applied Biosystems Inc., Austin, TX