Using Effective Controls for RNA Interference (RNAi) Experiments

siRNA-mediated RNAi has become an indispensable tool in functional genomics research.   Chemically synthesized siRNA reagents that target every gene in human, mouse and rat genome are available for convenient delivery in vitro.  Data acquired from RNAi experiments are used to support important conclusions about how genes function. 

How can you ensure that your conclusions are accurate?  A good way is to include proper controls in your experiments.  Controls not only help strengthen your conclusions, but also ensure that your RNAi experiments result in the expected silencing.  The importance of using appropriate experimental controls to maximize the value of your RNAi data is discussed in two recent publications [1,2].

How do you decide which controls are essential for your experiment?  Dharmacon recommends including at least three types of control samples in every RNAi experiment: positive control, negative control, and untreated control (Table 1).  Positive controls monitor efficiency of siRNA delivery into cells and negative controls distinguish sequence-specific silencing from non-specific effects.  Untreated samples determine the baseline level of cell viability, phenotype, and target gene level.  This Technical Note discusses the function and features of positive and negative control siRNAs.

Positive Control siRNAs

To achieve successful gene silencing, efficient siRNA delivery into cells is paramount.  siRNA delivery efficiency can vary between the cell types used and the delivery method used.  All delivery methods can produce a negative effect on cell viability.  A well-characterized positive control siRNA is an ideal reagent for establishing parameters that result in successful siRNA delivery without affecting cell viability.

An siRNA that targets a housekeeping gene ensures that the target gene is expressed in all cell types at a level that does not fluctuate with cell cycle.  An abundantly expressed gene allows easy and accurate assay of its mRNA and protein levels.  However, silencing of the gene should not affect either the cell phenotype or its viability.  Three popular target genes that meet these criteria are cyclophilin B (also known as PPIB), glyceraldehyde-3-phosphate dehydrogenase (GAPD), and Lamin. Dharmacon offers the siCONTROL® line of siRNA products (Table 1) to target these well characterized targets.

The ideal positive control siRNA is highly functional, as the level of silencing it produces needs to be correlated with the efficiency of delivery siRNA.  Dharmacon assigns a threshold of 80% cell viability and 75% gene silencing for successful delivery of siRNA.  As an example, Figure 1 shows the results for a transfection optimization experiment for 3 x 10⁵ HeLa cells in 6-well plates.  The positive control siRNA used in this experiment (siCONTROL Cyclophilin B siRNA) has been characterized to silence the target mRNA 85% under optimal conditions.  Therefore, the samples in which the silencing is lower than 85% were not optimally transfected.  From this experiment, we concluded that 4 or 6 µL DharmaFECT® 1 is best for this system, as they result in at least 85% silencing of cyclophilin B while maintaining at least 80% cell viability.

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Table 1: Recommended Controls for RNAi Experiments

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siRNA delivery efficiency may vary greatly from one experiment to another due to multiple factors such as variations in cell passage and batch of transfection reagent.  Therefore, Dharmacon recommends including a positive control in every RNAi experiment.  The silencing level and cell viability of the positive control sample allows confirmation that the siRNA delivery process in each experiment is successful and non-toxic.  Lower than expected silencing of the target gene indicates less than optimal delivery, but still allows normalization of the data to allow comparison with other experiments.  However, lower than 80% cell viability may indicate compromised cell viability and usability of the data from this experiment should be reassessed.

Figure 1.  Using a Positive Control siRNA to Optimize Transfection Efficiency.  Cyclophilin B mRNA level was measured in HeLa cells 24 hours after transfection with 100 nM siRNA targeting cyclophilin B or negative control siRNA using 0.5 – 6 µL/well DharmaFECT 1. Cyclophilin B mRNA level was measured using bDNA (Panomics (Freemont, CA)).  Cell viability was measured by alamarBlue™ assay (Biosource International (Camarillo, CA)).  All values are expressed as percent of untreated sample (mean ± SD, n=3).

Fluorescent-labeled RNAi controls

Fluorescent-labeled RNAi controls are popular as transfection efficiency controls as they provide a rapid visual evaluation.  The fluorescence may be detected by either fluorescence microscopy or flow cytometry.  Dharmacon offers two transfection indicators, siGLO Red and siGLO Green, to provide reliable visual assessment of transfection success. The fluorescent signal localizes to the nucleus as an unmistakable signal of efficient uptake, while the maximum fluorescence correlates to the optimal siRNA uptake conditions.

Negative Control siRNAs

Negative control siRNAs are designed to have no known target in the cells being used.  They are important for distinguishing sequence-specific silencing from non-specific effects in the RNAi experiment.  Samples that are treated with a negative control siRNA are analyzed similarly to samples that are treated with the siRNA targeting your test gene.  Neither the mRNA nor protein level of the experimental gene should be affected by the negative control siRNA.  For example, Figure 1 shows that samples treated with a negative control siRNA have comparable cyclophilin B mRNA level as the untreated sample.  The lack of cyclophilin B silencing by the negative control siRNA confirms that the silencing by the cyclophilin B siRNA is sequence-specific.

Cell viability as well as cell phenotype of samples treated with a negative control siRNA should also remain comparable to those of untreated samples.  When these conditions are met, it can be concluded that silencing by the test siRNA is specific, and any downstream effect to gene knockdown can be attributed to the specific silencing of the target gene.

There are a variety of negative control siRNAs commercially available.  The most common group of negative control siRNAs are designed to have no known mRNA targets in the cells used, and are described as non-targeting siRNAs.  Dharmacon offers many options for negative control siRNAs, including the ON-TARGETplus siCONTROL Non-Targeting siRNA (Table 1). Recent characterization of the microRNA seed region, nucleotides 2 – 6 of the siRNA antisense strand, predicts that nucleotide identity of this region may allow the siRNA to act through the microRNA pathway.  Thus, a negative control siRNA should not only have minimal overall sequence identity between the negative control siRNA and any known gene, but should especially have no sequence identity between the miRNA seed region and any known gene in the cell.  Consequently, an siRNA with only a few nucleotide mismatches to the target mRNA that are considered essential for siRNA binding and activity may not serve as a good negative control.  Thus, the ON-TARGETplus siCONTROL Non-Targeting siRNA is not only designed and chemically-modified to not target known genes in human, mouse and rat cells, but microarray-confirmed to have no detectable silencing in human cells.

Another negative control strategy is the "scramble" siRNA that has the same nucleotide composition, but not the same sequence, as the test siRNA.  This is achieved in two ways: randomizing (also known as scrambling) the nucleotides in the siRNA or reversing the sequence of the siRNA.  When their sequences are confirmed to not target a known gene or have an miRNA seed region match with a known gene, these siRNAs are appropriate as negative control siRNAs.  However, it is not practical and often impossible, to identify a scramble control for every gene-specific siRNA that is used, therefore a universal non-targeting control is more practical. Dharmacon offers several options for universal non-targeting control siRNAs, including the ON-TARGETplus siCONTROL Non-Targeting siRNA (Table 1).

Other Control siRNAs

Chemically modified control siRNAs

Does a control siRNA need to have the same modification as the test siRNA?  Possibly.  Generally, a control siRNA should be as similar to the test siRNA as possible.  For example, if the test siRNA is chemically modified to improve specificity of target-binding, then the control siRNA could also have the same chemical modification.  This allows us to conclude that effects observed with the test siRNA are due to specific silencing of the target gene, and not due to non-specific effects related to the chemical modification.  Dharmacon offers two different sets of chemically modified control siRNAs suitable for use with similarly-modified experimental siRNAs, siSTABLE® and ON-TARGETplus. siSTABLE control reagents are stability-enhanced to to extend siRNA stability in nuclease-rich environments while the ON-TARGETplus control siRNAs are chemically modified for greater specificity.

Pooled control siRNAs

Does a negative control siRNA need to be pooled when multiple experimental siRNAs are pooled for silencing?  Not necessarily.  Dharmacon recommends using pooled siRNAs as a single reagent.  As such, the appropriate control may be either a single siRNA or pooled siRNAs.  However, it is important to use the same negative control siRNA concentration as the concentration of the experimental siRNA. Dharmacon offers several pooled control reagents inlcuding the positive control reagents ON-TARGETplus siCONTROL Cyclophilin B Pool, and ON-TARGETplus siCONTROL GAPD Pool, and the negative control ON-TARGETplus siCONTROL Non-Targeting Pool.

Dharmacon Solutions

Dharmacon offers a wide selection of positive and negative control siRNAs to meet many experimental setups (Figure 2).  They are available unmodified or chemically modified to improve targeting specificity (ON-TARGETplus siCONTROL siRNAs) or nuclease stability (siSTABLE siRNAs) for use with test siRNA with similar modifications (Figure 2).  For qualitative estimation of siRNA uptake, we also offer fluorescent-labeled siRNAs (siGLO).  

Visit www.dharmacon.com for more information and to purchase the controls that are right for you.

Figure 2. Choosing Control siRNAs
(a larger version of the flow diagram can be viewed by going here.)

References Editorial. "Whither RNAi?" Nat Cell Biol June 1, 2003 2003: 489-90. Huppi, K., S. Martin, and N. Caplen. "Defining and Assaying RNAi in Mammalian Cells." Mol Cell 17.1 (2005): 1-10.

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