SureSilencing shRNA Plasmids
For knockdown of human, mouse, or rat gene expression by RNA interference
SureSilencing shRNA Plasmids are available for every gene in the human, mouse, and rat genome. For each gene, a set of 4 shRNA plasmids are provided, each targeting a different section of the gene, to knock down the expression of the target gene using short-hairpin RNA.
SureSilencing shRNA Plasmids specifically knock down the expression of every human, mouse, or rat gene by RNA interference. For each gene, 4 separate shRNA designs are provided, packaged in a plasmid backbone.
The experimentally verified shRNA design algorithm ensures maximum gene specificity and RNAi efficacy. Greater than 70% knockdown for the targeted gene will be observed with at least 2 of the 4 shRNA plasmids (as measured by real-time RT-PCR in transfected cells upon FACS-based enrichment for GFP expression or selection for antibiotic resistance) (see figure "Knockdown of gene expression by at least 70%"). The availability of 2 effective sequences controls for nonspecific and off-target effects.
Short-hairpin RNA (shRNA), one method of enabling RNA interference (RNAi), uses a plasmid- or viral- based expression vector, delivered to a cell population by transfection. The short RNA sequence expressed by the vector folds into a short hairpin and is processed by the cellular RNAi machinery. SureSilencing shRNA Plasmids use a plasmid-based expression vector to enable RNAi.
SureSilencing shRNA Plasmids are available in 4 different plasmid formats. Plasmids contain either the hygromycin, neomycin, or puromycin resistance gene or GFP.
Plasmids containing the hygromycin, neomycin, or puromycin resistance gene enable selection of stably transfected cells and are well suited for the study of the long-term effects of gene knockdown in cells. Plasmids containing the GFP gene offer an alternative to siRNA, enabling the identification and tracking of transiently transfected cells. They are highly suited to the study of short-term gene knockdown effects. The ability to track or stably express shRNA enables RNAi in difficult-to-transfect cells.
Unlike chemically synthesized siRNA, plasmid-based shRNA provides a renewable source of RNAi. Enough plasmid can be amplified in the lab to complete a project. SureSilencing shRNA Plasmids also include complete shRNA sequence information.
SureSilencing shRNA Plasmids are first transformed into competent E. coli cells. Colonies are picked for large-scale bacterial culture and large-scale, high-quality transfection-grade plasmid preparation. Gene-specific and negative control shRNA plasmids are transfected into replicate wells of the cell line of interest and incubated for 24–48 hours.
Following transfection with a GFP SureSilencing shRNA Plasmid, cells can be tracked and characterized using fluorescence microscopy or enriched using fluorescence-activated cell sorting (FACS) or antibiotic selection. Following transfection with a hygromycin, neomycin, or puromycin SureSilencing shRNA Plasmid, cells may then be replated into medium containing the appropriate antibiotic to begin the process of selecting for and cloning stably transfected cells. The extent of knockdown can be verified using RT2 Profiler PCR Arrays or RT2 qPCR Primer Assays.
SureSilencing shRNA Plasmids are highly suited for gene function studies, removal of protein activity, and signaling pathway studies.
fragment fix placeholder