RNAI: TIPS TO BETTER SILENCING
A few general rules make it relatively easy to knockdown genes with RNA interference (RNAi). In general, the process introduces a short interfering RNA (siRNA) into a cell. The siRNA gets incorporated into the RNA-induced silencing complex (RISC), which targets the desired transcript for degradation. The complexity comes in the source of the siRNA. Moreover, the various sources of siRNA work a bit differently.
Today, most scientists purchase ready-to-use RNA. Nonetheless, it’s still worth keeping in mind the following points:
1.Use More Than One Sequence
For scientists making siRNAs from scratch, try several siRNAs for every gene targeted for knockdown. Even when purchasing siRNA designed to knockdown a specific gene, try more than one, because RNAi can lead to false positives and false negatives. By using multiple siRNAs, consistent outcomes distinguish on-target effects.
2.Make the Right Modifications
Some researchers chemically modify their siRNAs for greater stability or tissue targeting. This can increase the odds that an siRNA can survive its journey to the right cell. Some modifications, such as replacing ribouracil with ribothymine, can also reduce off-target effects. Most RNAi vendors sell modified and unmodified siRNA, but modifications can significantly increase the cost.
3.Consider Aiming at DICER
Although most companies provide siRNA that binds to RISC, some companies sell DICER-substrate RNAi. These molecules are longer than siRNA and also double-stranded. The Dicer enzyme converts a long duplex RNA into siRNA. The Dicer-substrates offer pros and cons, such as potentially higher knock down in some cases and less stability where the long duplex RNA must travel through body fluids to a cell. To provide more stability, some companies encase the Dicer-substrate in liposomes.
4. Optimize Silencing with Vectors
Longer-lasting effects can be achieved with a DNA-based vector system that expresses short hairpin RNAs (shRNAs). The shRNAs typically contain stem structures 19–29 nucleotides in length, capped with a five-to-nine base loop sequence. The shRNA approach also works well for cells that are difficult to transfect. Like siRNA, the shRNA incorporates with RISC to turn on RNAi. The shRNA can also be purchased from many vendors.
