Recommended Controls and How to Accomplish - Click for larger version
Scrambled (Negative) siRNA Control
Comparing cells transfected with a nonsense or scrambled sequence siRNA control to nontransfected cells can reveal changes caused by siRNA delivery. Silencer Negative siRNA Control #1 is a validated negative control that has limited sequence similarity to the human, mouse, or rat genomes. Ambion scientists, as well as many other scientists around the world, routinely use this negative control in their RNAi experiments.
Positive siRNA Control
A positive siRNA control should be used in experiments to monitor and optimize siRNA transfection efficiency. When the positive control fails to elicit the expected reduction in gene expression, poor transfection is immediately suspected. Ambion's Silencer GAPDH siRNA is a commonly used positive control because GAPDH is expressed at easily detectable levels in most cell types.
Multiple siRNAs to a Single Target
There are several example s of single siRNAs affecting the expression of multiple genes. This unwanted experimental result is explained by non-specific "off-target" effects. Controlling for siRNA specificity involves simply using multiple siRNAs targeting different regions of the transcript being studied. The siRNAs should first be analyzed for effective reduction of target gene expression. Different siRNAs to the same target and with comparable gene silencing efficacy (i.e. mRNA reduction) should induce similar phenotypic effects. Any changes induced by one siRNA and not the other(s) could be attributed to off-target effects. One of the easiest ways to obtain multiple effective siRNAs to the same target is by searching Ambion's database of Silencer Pre-designed siRNAs. When you purchase three Silencer Pre-designed siRNAs to the same target, Ambion will guarantee that at least two of those siRNAs will knockdown target mRNA levels by 70% or greater.
Monitor Both Target mRNA and Protein Levels
siRNAs trigger mRNA degradation through the RNAi pathway. In contrast, the closely related microRNAs (miRNA) usually inhibit translation without significantly affecting mRNA levels (see MicroRNAs: Isolation to Functional Analyses, for more information). M ost researchers agree that it is important to monitor RNAi induced knockdown at both the mRNA and protein levels. With an effective siRNA, you would expect to see reduction of both target mRNA and corresponding protein levels. mRNA reduction seen without a corresponding reduction in protein levels can indicate that protein turnover is slow--a different time point may be required for your biological assay. In contrast, protein reduction in the absence of mRNA reduction may indicate that an siRNA is mediating its effects at the translational level as an miRNA. To save time and conserve reagents, you can analyze mRNA and protein levels from a single sample. The PARIS and mirVana PARIS Kits, which provide a simple protocol to isolate native protein and total RNA from the same sample, makes this particularly straightforward.
Use the Lowest Possible siRNA Concentration
Several reports indicate that siRNA concentrations of 100 nM or higher in mammalian cultured cells can lead to nonspecific changes in gene expression [2, 3]. Using 5-20 nM of a highly effective siRNA appears to minimize nonspecific effects. Well-designed siRNAs, such as Ambion's Silencer Validated and Pre-designed siRNAs, can often be used at even lower concentrations, further reducing the chance of eliciting off-target effects. Figure 1 shows a titration experiment in which various concentrations of Silencer 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 RNAi effect is reversed through expression of a target gene refractory to silencing by a particular siRNA can give final confirmation that an observed effect is specifically due to knocking down the target of interest. Like array analysis, this experiment is time consuming.
While considered the ultimate controls, editors of Nature Cell Biology and other journals do not require these experiments for publication in their journals.
Controls for RNAi Screening Experiments
Use of many of the above controls in large scale RNAi screens is virtually impossible. At a minimum, one or more negative and positive control siRNAs should be used per plate analyzed. And validation of some or all of the "hits" using more extensive controls is warranted. Finally, the best way to improve confidence in RNAi data is to use at least three siRNAs in independent wells of the screen. See Setting up Successful siRNA Library Screens, for more information about ways to use siRNA libraries for both large and small scale RNAi screens.
As the RNAi Field Evolves
The use of RNAi has enormous potential for analyzing gene function, elucidating biological pathways, and identifying and validating potential drug targets. The RNAi field is still relatively new, and recommendations for experimental design and proper c ontrols are likely to evolve. Ambion is committed to bringing researchers around the world the most up-to-date information on RNAi for their research. For the latest information, see the RNAi Resource Page at www.ambion.com/RNAi.
Cat# Product Name Size 1552 mirVana miRNA Detection Kit 100 rxns 1556 mirVana PARIS Kit up to 40 purifications 1751 MessageAmp II aRNA Amplification Kit 20 rxns 1921 PARIS Kit 50 purifications 1924 RiboPure Kit 50 purifications 4300 anti-GAPDH, mouse monoclonal 6C5 100 g 4605 Silencer GAPDH siRNA (Human ) 5 nmol + 2 nmol Neg Control (50M) 4611 Silencer Negative Control #1 siRNA 5 nmol (50 M)