How many shRNAs should be tested for knocking down a gene of interest?
When using shRNAs, it is important to recognize the fact that not all shRNAs will work. Knockdown effects of empirically designed shRNAs are often limited by variations in specificity and efficiency observed from one shRNA to another. The potency of an shRNA is determined by several factors including length of the shRNA, loop structure, GC profile and thermodynamic stability of the shRNA, secondary structure of the target sequence and off-target matches to other genes. Typically, ~50-70% of shRNAs have noticeable knockdown effect, and ~20-30% of them have strong knockdown. Therefore, it is important to test multiple shRNAs to find the most potent shRNA for knocking down your gene of interest.
Based on our experience and feedback from our customers, we know that generally when 3 or 4 shRNAs are tested for any arbitrary gene, typically 2 or 3 produce reasonable to good knockdown. If you try a few shRNAs targeting a specific gene, it is possible that by chance, none will produce satisfactory knockdown. When this happens, the best approach is to try more shRNAs, especially the ones that have literature validation. Many researchers also use a “cocktail” of shRNAs (i.e. mixture of different shRNAs) targeting the same gene, which sometimes can improve knockdown efficiency.
VectorBuilder has created shRNA databases that contain optimized shRNAs for common species. shRNAs in these databases are designed and scored applying rules like those used by the RNAi consortium. When you design shRNA vectors using our online design tool, you will have the option to search your target genes in our database. Once you enter your genes of interest, you will see detailed information of all available shRNAs we have designed for you, including a link to UCSC Genome Browser to view these shRNAs in the context of their genomic sequence and all the transcript isoforms. Our database ranks all available shRNAs for a target gene in order of their decreasing knockdown scores and recommends testing the top 3 shRNAs with the highest knockdown scores.
Lentiviral vectors are the most preferred shRNA delivery vehicle for applications requiring long-term knockdown of gene expression. However, several researchers prefer to use regular plasmid vectors for transient transfection and validation of candidate shRNAs targeting their genes of interest to minimize higher costs and technical complexities associated with lentiviral vector-based methods. Once the shRNA(s) with the highest knockdown efficiency is identified, it can then be expressed using a lentiviral vector for achieving stable knockdown of the target gene.