Loading ...RNA interference (RNAi) is a valuable tool used by molecular biologists to produce genetic knockouts and elucidate the consequences of mutations in individual genes. Despite numerous advances in RNAi methods and the accumulation of a substantial body of peer-reviewed publications demonstrating the power and versatility of these methods, many researchers continue to find that conducting RNAi experiments is a challenging process and that interpreting their data is complicated by false negatives and generalized effects which are not accounted for in their experimental designs. Here are a few tips for how to design RNAi experiments that avoid these problems.
Chose your sequence carefully
Choose at least four sequences for each target, usually 20-35 bases long, that begin with a low wobble series, such as AA, or AC/CA (not CC), and have no more than 55% GC content with GC residues evenly spaced throughout (clumps of GC’s can lead to a loss of specificity).
Use the equation Tm= 4 x (G,C) + 2 x (A,T) ̊C to chose sequence with melting temperatures 5-10 degrees higher than your experimental conditions as this allows for more complete annealing to your target.
Make sure that your sequences are not self-complementary (palindromic), and that they will not form hair-pins (unless your are using a Dicer protocol).
BLAST your sequences against the genome you are targeting. A carefully targeted sequence is specific for your mRNA target. If your genome hasn’t been sequenced to BLAST against all genomes and use your best judgment to select several sequences for your experiment.
Prepare your reagents carefully
Filter your RNA to remove salts, proteins, fragments, and loose nucleotides. Glass filters and elution or gel purification are the most common routes.
Some protocols call for chemical modification of your sequences (U-> T , for instance). These modifications are usually not essential, but can improve results, however, if you modify or synthesize your own RNA’s then you will probably need to purify by gel electrophoresis.
Take care to avoid exposing your reagents to RNAases. These proteins will destroy your probes, and it can be difficult to prevent contamination.
Don’t use antibiotics until after transfection is complete as antibiotics enter cells during transfection and can accumulate to toxic levels. It is a good rule to wait at least 48 hours, although some sources recommend that 72 hours be allowed before application.
Use the right transfection agents. RNAi probes are not plasmids. Use specially prepared reagents for transfection in RNAi experiments to prevent high levels of probes from accumulating in cells as this can lead to toxicity and cell death. Generally, lower concentrations or pore-inducing salts are required for RNAi protocols.
Design your experiment well
Perform several initial experiments with different probes, using different concentrations of your probes and transfection buffers. Evaluate the results to optimize your protocol and repeat to test their reproducibility.
Use florescent or radio tags attached to your probes to evaluate the efficiency of your transfection, the persistence of your probe in the target cell, and the localization of your probe within these cells. If your probe isn’t entering the cell, is being degraded, or is binding to something it shouldn’t be this can be a good way of finding out.
Utilize a positive and negative control. Housekeeping genes make good positive controls. Targeting these during the optimization described above, and checking their expression levels 48 hours latter can provide valuable information about the efficiency of your transfections. In contrast, using a randomized probe sequence that lacks homology to the target genome, but is identical in length and raw base composition to your experimental probe provides an indication of whether off-taget effects and general toxicity are an issue. If your cells aren’t showing any response to your protocol or they are dying then these controls may help you understand why.
Let software help you
MiraiBio’s free DNASIS SmartNote web application includes an siRNA tool called DEQOR, designed to help you design RNA interference experiments. Its advanced algorithms score siRNAs based on sequence length, GC-content, nucleotide composition and cross-silencing ability. It only takes a minute to sign up. Try it out today!
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