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Negative controls that do not target any endogenous transcript are needed to control for non-specific effects on gene expression caused by introducing any siRNA. Easy-to-assay positive controls are needed to optimize transfection conditions, ensure that siRNAs are delivered, and ascertain that a particular downstream assay is working. Because positive controls are required for many different aspects of an RNAi experiment, often more than one control is required. Ambion scientists use and recommend a number of different controls for siRNA experiments. Most of these coincide with the suggested controls detailed in an editorial published in Nature Cell Biology [Whither RNAi? (2003) Nature Cell Biology 5: 489–490]. |
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Recommended Control |
How to accomplish |
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1. Negative Controls: Nonspecific or scrambled siRNAs |
Use Silencer Negative Control #1 (#4611) •Limited sequence similarity to human, mouse, and rat sequences•Verified as a control by extensive experiments, including array experiments |
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2. Positive, validated siRNA control: Assay target mRNA levels to monitor transfection efficiency and RNAi induction |
Use Silencer GAPDH siRNA Control (#4605) to verify transfection efficiency and induction of RNAi •Extensively validated in human cells)•Easy to assay target •Corresponding antibody available (Cat#4300) |
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3.Multiple siRNAs to same target: Best way to improve confidence in RNAi data and control for sequence-specific off-target effects |
Use Silencer Pre-designed siRNAs •Three siRNAs available for most targets•Guaranteed silencing – When 3 siRNAs purchased, at least 2 guaranteed to silence target mRNA ≥70% •Available to >34,000 human, mouse and rat targets |
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4.Titration of the siRNA: Use at the lowest effective level |
Use Silencer Validated and/or Pre-designed siRNAs at low concentrations •Designed for maximum potency•siRNAs typically effective at ≤10 nM |
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5.Monitor both mRNA and protein levels |
Isolate protein and RNA from the same sample with the PARIS Kit •Simple protocol•Save time and reduce variability by using same sample for both protein and RNA analysis |
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6.Monitor off target effects and antiviral response |
For the most thorough analysis, perform array analysis. •RiboPURE Kit for isolating array quality RNA•MessageAmp II aRNA Amplification Kit for ultra high yields of amplified cRNA |
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7.Rescue experiments: Introduce construct expressing target mRNA refractory to RNAi |
As part of rescue experiment, monitor mRNA and siRNA levels with the mirVana miRNA Detection Kit •Sensitive detection•Quantitate siRNA and target mRNA in same sample |