RNA interference is an effective mechanism for gene silencing whereby double-stranded RNA triggers the cleavage and subsequent degradation of homologous transcript sequences. In this evolutionarily conserved process, longer double-stranded RNA molecules are processed into shorter sequences (21–23 nucleotide small interfering RNAs, or siRNAs) that can bind to the multicomponent RNA-induced silencing complex (RISC).
Within this complex, the siRNA is unwound, and the sense strand is cleaved and dissociated. The antisense strand then remains bound and acts as a guide to target activated RISC to complementary mRNA for cleavage and degradation.
This potent, sequence-specific RNA degradation mechanism was first discovered in plants, where it was termed post-transcriptional gene silencing. It has since been demonstrated in a wide variety of eukaryotic organisms, ranging from fission yeast to humans. These discoveries and subsequent studies into how the RNAi process works have enabled researchers to exploit this pathway and develop tools in order to better elucidate gene function.
Plasmid-based expression of gene-specific small hairpin RNAs (shRNA) under the control of RNA polymerase III–dependent promoters is an effective way to trigger this process. With this approach, the shRNAs are processed intracellularly by the enzyme Dicer into siRNAs, which are then able to directly engage RISC.
The Mission TRC1 shRNA libraries from Sigma-Aldrich consist of over 150,000 such plasmid-based shRNA constructs targeting 15,000+ human and 15,000+ mouse genes. The shRNA sequences are designed using an algorithm developed by the Broad Institute of MIT and Harvard.
On average, there are five shRNA designs for each gene target. The shRNA plasmids are further processed into lentiviral particles to facilitate stable gene silencing in both dividing and quiescent cells. The LentiPlex libraries were generated from the RNAi Consortium’s TRC1 library and are intended for rapid, whole-genome, pooled RNAi screening projects.
While individual genes can be efficiently and robustly targeted using arrayed lentiviral libraries, pooled shRNA libraries may be used to rapidly conduct many phenotypic screens. These pooled screens are typically set up such that the majority of cells have been transduced with a single shRNA to aid in downstream deconvolution.
Positive selection screens are an example of the type of screen that may be conducted using pooled libraries. Selection of a desired phenotype is the basis of this type of screen where cell viability/survival is a commonly used phenotype. After selection, integrated shRNAs are identified by traditional Sanger or deep sequencing methods. The identity of potential hits can then be used to develop hypotheses regarding the biological role of the corresponding gene(s). As with all screens, validation of leads by independent methods such as siRNA, small molecule inhibition, or gene knockout will be required.
The Mission LentiPlex Human Pooled shRNA Library and the Mission LentiPlex Mouse Pooled shRNA Library are genome-wide lentiviral pools. Representation of individual shRNAs from each library is tested before product release to ensure robust library coverage. The actual number of clones in each subpool may vary. Each library is provided as 2 x ~25 µL aliquots of ready-to-use lentiviral format at titers of at least 5 x 108 TU/mL via p24 assay and is predivided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream hit identification.