Why isn't my shRNA knocking down my gene of interest?
Not all shRNAs will work
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. However, when using shRNAs, it is important to recognize the fact that not all shRNAs will work. Typically, ~50-70% of shRNAs have noticeable knockdown effect, and ~20-30% of them have strong 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.
The assay for validating the knockdown of your gene is not performed properly
The most common and sensitive assay to evaluate shRNA knockdown efficiency is RT-qPCR. Sometimes, you may need to try several pairs of primers, and then choose the most specific and efficient pair to use. In general, the RT-qPCR primers should span exon-exon junction if possible to avoid amplifying genomic DNA. When using a new pair of primers, we recommend that you run the PCR product on an agarose gel to verify the band, or even validate the PCR product by sequencing. You should always include minus-RT control in RT-qPCR to better estimate the level of genomic DNA contamination. You can use NCBI primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to help you better examine the quality of your primers in silico.
Knockdown efficiency can also be assessed by Western blot. However, Western blot is notoriously prone to false positive bands from non-specific antibody binding, which could mistakenly lead to the interpretation that there is no knockdown. Care must therefore be taken to make sure that the antibody used is indeed specific to the gene of interest.
The shRNA might only target a subset of transcript isoforms of your gene
When designing shRNA, we generally recommend those that can target as many transcript isoforms of the gene as possible, unless you are only interested in knocking down a particular isoform. VectorBuilder has created shRNA databases that contain optimized shRNAs for common species. If you design shRNA vectors on VectorBuilder, when you insert the shRNA component into the vector, you will have the option to search the target gene in our database. Then, you will see the detailed information of all the available shRNAs we designed for you, including a link to UCSC Genome Browser to view these shRNAs in the context of genomic sequence and all the transcript isoforms.