Navigation Links
Designing Controls for siRNA ,,, Experiments

Because of its apparent specificity, reproducibility, and ease of use, RNAi technology is greatly accelerating the functional characterization of disease-relevant genes for drug discovery, target validation, and basic research efforts. To ensure the validity of RNAi data, however, proper experimental design and controls are needed. This article outlines some of the critical parameters for performing RNAi experiments in mammalian cultured cells.

Tips for Experimental Design

Carefully Choose Your siRNA Sequence for Maximum Specificity.
Although data indicate that siRNAs are highly specific, it is important to minimize the possibility of off-target effects by designing siRNAs that have limited sequence similarity to genes other than their intended target. Currently, it is not clearly understood how the number and location of mismatches between the antisense siRNA strand and the target mRNA affect silencing. Some reports indicate that a single mismatch in the center of an siRNA sequence can abolish silencing effects (1,2). In fact, this remarkable specificity has been shown in a model system to permit allele specific gene silencing (2). In contrast, another report indicates that siRNAs with regions of 14-15 contiguous bases of sequence similarity between the siRNA and an mRNA can induce silencing (3). Until we develop a better understanding of the specificity of siRNAs for their targets, it is best to design siRNAs that are completely complementary to the mRNA target and that contain at least 2 or more mismatches to all off target mRNAs. Also, results obtained with pools or populations of siRNAs should be confirmed with individual siRNAs to enhance the confidence level in the data.

Use the Minimum siRNA Concentration Required to Achieve Silencing.
Recent reports indicate that siRNA concentrations of 100 nM or higher in mammalian cultured cells can lead to nonspecific changes in gene expression (3,4). In general, transfecting 5-20 nM of an efficacious siRNA appears to minimize nonspecific effects while providing maximal target gene silencing. The concentration of siRNA required will depend upon the method and efficiency of siRNA delivery, the cell line used, and the effectiveness of the siRNA sequence.

Suggested Experimental Controls

Ambion scientists use and recommend a number of different controls for siRNA experiments. Most of these coincide with the suggested controls detailed in a recent editorial published in Nature Cell Biology (5). These controls can be used in virtually any gene silencing experiment, whether the effects are analyzed one gene at a time using more traditional mRNA and protein analysis techniques or on a genome-wide scale using microarrays.

Scrambled siRNA Control
A scrambled siRNA control is used to discount any ch anges to the gene expression profile that may result from the siRNA delivery method. Comparing cells transfected with a scrambled siRNA control to untransfected cells can reveal changes caused by siRNA delivery.

Positive siRNA Control
Ambion scientists always include a positive siRNA control in their experiments -- usually the Silencer GAPDH siRNA -- which serves to monitor siRNA transfection efficiency. When the positive control fails to elicit the expected reduction in gene expression, poor transfection is immediately suspected.

Multiple siRNAs to a Single Target
One of the best ways to increase confidence in data from siRNA experiments is to use two or more siRNAs to a single gene target. The siRNAs should first be analyzed for effectiveness at reducing target gene expression. Different siRNAs to the same gene with comparable gene silencing efficacy should induce similar changes in gene expression profile and phenotype. Any changes induced by one siRNA and not the other(s) could be attributed to off target effects. This approach is also valid for siRNA cocktails. In other words, cocktails prepared from different regions of the same gene can be used to confirm gene silencing results.

Monitor the Antiviral Response
Recent evidence indicat es that up-regulation of the antiviral response may be a useful indicator of nonspecific siRNA effects. Although we do not yet routinely monitor the antiviral response in our own experiments, there are several published reports on how this response can be detected. One way is to assess levels of 2'5'-oligoadenylate synthetase mRNA (6). 2'5' oligoadenylate synthetase catalyzes the synthesis of 2'-5' polyadenylic acid, which activates the nonspecific ribonuclease RNase L. Another way is to monitor activation of the dsRNA-dependent protein kinase, PKR, which is involved in the type 1 interferon response. PKR phosphorylates the small subunit of the eukaryotic initiation factor 2-alpha (eIF2alpha), which results in nonspecific inhibition of translation. PKR activation can therefore be examined by monitoring the phosphorylation of eIF2alpha (7).

Monitor Both Target mRNA and Protein Levels
In siRNA experiments it may be beneficial to monitor both mRNA and protein levels for several reasons. For instance, mRNA reduction seen without a corresponding reduction in protein levels can indicate that protein turnover is slow. Furthermore, protein reduction in the absence of mRNA reduction may indicate that an siRNA is mediating its effects at the translational level like a microRNA.

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 in its infancy, and recommendations on experimental design and proper controls are likely to evolve. Ambion is committed to bringing researchers around the world the most up-to-date information on RNAi for their own research. For the latest information, see the RNAi Resource.

back to top



Page: All 1 2 3 4 5

Related biology technology :

1. Designing a Successful qRT-PCR Experiment
2. Designing a Better siRNA
3. Housekeeping Genes: Universal Positive Controls in siRNA Knockdown Experiments
4. RNAi: The Controls You Need
5. Custom and library siRNA for efficient gene silencing
6. Custom and library siRNA for efficient gene silencing
7. Cancer siRNA Oligo Set Version 1.0
8. Library siRNA
9. Custom siRNA Oligo Synthesis Service
10. Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and TransMessenger Transfection Reagent*
11. Quantification of siRNA Silencing Efficiency Using the LightCycler System
Post Your Comments:

(Date:11/30/2015)... HOLLISTON, Mass. , Nov. 30, 2015 /PRNewswire/ ... HART ), a biotechnology company developing bioengineered ... has received written notification from The NASDAQ Stock ... minimum bid price requirements. The letter noted that ... of HART,s common stock having exceeded $1.00 per ...
(Date:11/30/2015)... DIEGO , Nov. 30, 2015 Human ... that the company has acquired Cypher Genomics, Inc., a ... robust human genomic interpretation software solutions. The ... will join HLI including Cypher CEO and Co-founder, Ashley ... of HLI,s Pediatric Business.  Financial details of the deal ...
(Date:11/30/2015)... and MAGDEBURG, Germany , November ... NeuroRehabilitation (ECNR) in Vienna, Austria ... European Congress of NeuroRehabilitation (ECNR) in Vienna, ... --> NovaVision, a wholly owned subsidiary of Vycor Medical, ... version of its Internet-delivered NovaVision Therapy Suite at the ...
(Date:11/27/2015)... United Kingdom , Nov. 27, 2015 /PRNewswire/--  Mallinckrodt ... biopharmaceutical company, announced today that it has closed the ... (CMDS) business to Guerbet (GBT- NYSE Euronext) in a ... operations encompassed four manufacturing facilities and a total of ... 75 in the St. Louis ...
Breaking Biology Technology:
(Date:11/4/2015)... ALBANY, New York , November 4, 2015 /PRNewswire/ ... According to a new market report published by Transparency ... Size, Share, Growth, Trends and Forecast 2015 - 2022", ... value of US$ 30.3 bn by 2022. The market ... during the forecast period from 2015 to 2022. Rising ...
(Date:10/29/2015)... 29, 2015  Rubicon Genomics, Inc., today announced ... of its DNA library preparation products, including the ... ThruPLEX Plasma-seq kit. ThruPLEX Plasma-seq has been optimized ... NGS libraries for liquid biopsies--the analysis of cell-free ... applications in cancer and other conditions. Eurofins Scientific ...
(Date:10/27/2015)... -- Munich, Germany , October ... automatically maps data from mobile eye tracking videos created ... that they can be quantitatively analyzed with SMI,s analysis ... , October 28-29, 2015. SMI,s Automated Semantic Gaze ... tracking videos created with SMI,s Eye Tracking Glasses ...
Breaking Biology News(10 mins):