RNA interference (RNAi) is the process of mRNA degradation that is induced by double-stranded RNA (dsRNA) in a sequence-specific manner. RNAi plays a vital role in sequence-specific cellular responses to RNA, collectively called RNA silencing. These responses have been shown to play a role not only in mRNA and dsRNA stability/degradation, but also in the regulation of transcription and translation, chromatin structure, and genome integrity. In all RNA silencing pathways, dsRNA is processed to 21–30 nucleotide (short) interfering RNA molecules (siRNAs). These siRNAs are highly effective tools for specifically silencing the expression of a particular gene. By introducing double-stranded siRNAs specific to a particular gene transcript, one can specifically disrupt the function of that gene. The power and utility of RNAi for specifically silencing gene expression has driven its incredibly rapid adoption as a tool for reverse genetics in eukaryotic systems.
RNAi experiments have four basic requirements: 1) an effectively designed RNAi molecule, 2) an efficient delivery method for the RNAi molecule, 3) a robust phenotypic assay for quantitative measurement of the RNAi effect, 4) positive and negative controls. A major challenge in siRNA research has been target-specific gene knockdown with minimum off-target gene modulation. Selecting the right homologous region within the gene is a very critical step. Factors such as the distance from the start codon, the G/C content and the location of adenosine dimers are important when considering the generation of dsRNA molecules for RNAi. Currently there are a number of different ways to silence gene expression using RNAi, depending on the model system being studied. In mammalian cultured cells, RNAi is typically induced by siRNA for transient knockdown, or a short hairpin RNA (shRNA) for stable integration. In some cases, siRNAs can be chemically synthesized or transcribed in vitro and then transfected into cells, injected into mice, or introduced into plants. In other systems, siRNAs can be expressed endogenously from siRNA expression vectors or PCR products in cells or in transgenic animals.
Invitrogen offers a very user friendly web based tool called BLOCK-iT™ RNAi Designer which can be used to design custom RNAi sequences for any organism, using a cDNA sequence or a GenBank accession number. The BLOCK-iT™ RNAi Designer, utilizes a highly effective, proprietary algorithm and can be used for efficiently designing different kinds of RNAi molecules for e.g., siRNA shRNA, miR RNA and Stealth™ RNAi sequences. It is such an effective tool that if you order the three to five best siRNA sequences designed, at least one or two of them will give greater than 70% knockdown of mRNA, as long as the transfection efficiency in your experiment is at least 80%.
Personally, I used Block-iT RNAi designer for designing shRNA molecules for cell cycle regulatory proteins. shRNA is a DNA molecule that can be cloned into expression vectors to express siRNA (19-21nt RNA duplex) for RNAi interference studies. shRNA has the following structural features: a short nucleotide sequence ranging from 19-29 nucleotides derived from the target gene, a short spacer of 4-15 nucleotides (i.e. the loop), and a 19-29 nucleotide sequence that is the reverse complement of the initial target sequence. The BLOCK-iT™ RNAi Designer takes care of all these structural features along with proprietary design criteria; you just need to input the sequence or accession number for your gene of interest. RNAi Designer will find and report 21 nt sequences for up to 10 top scoring gene-specific target sequences after BLAST comparison of input sequence with sequences in the database to find unique regions. You can either use a default loop or enter a custom loop sequence. RNAi Designer will add the appropriate linker sequences for directional cloning into Invitrogen's BLOCK-iTTM entry vector (pENTRTM/U6) vector or BLOCK-iT™ H1/TO vector. And then it will design top and bottom strands of the shRNA oligo by incorporating linker, target, loop and reverse complement of the target sequence. Finally, it will report to you the top strand, bottom strand and the annealed oligo sequence. You need to order the top and bottom strand sequences separately from any custom primer designing company (e.g. Invitrogen). An addition advantage of this tool is that you can easily convert already known or tested Stealth™ RNAi or siRNA sequences to shRNA sequences. However, BLOCK-iT™ RNAi Designer is specifically designed for Invitrogen RNAi vectors. If you decide to use cloning and expression vectors other than Invitrogen’s, you can still use the program, but you may need to add linker sequences manually according to your vector requirements. This program is helpful in reporting the 10 best RNAi sequences for the gene of interest; these sequences have an improved probability over random picking of inducing target gene silencing. However, a lot still depends on experimental design and conditions for efficient RNAi knockdown.
Finally, I find BLOCK-iT™ RNAi Designer as a very useful tool. Though these days lots of pre-tested shRNA libraries are available from various companies for a wide range of gene sequences. But if you choose to design your own RNAi molecules, the easiest way to proceed is BLOCK-iT™ RNAi Designer.
Post-Doctoral Fellow
Oregon Health Sciences University