http://www.genecopoeia.com/product/shrna/
OmicsLink™ shRNA clone collections consist of lentiviral, and other mammalian expression vector–based small hairpin RNA (shRNA) clones against genome-wide target genes from human and mouse. A set of four expression constructs for every target gene ensures high knockdown efficiency with minimal off-target effects.
GeneCopoeia provides four shRNA constructs for every target gene and guarantees that at least one of the four will have a knockdown effect of 70% on corresponding gene expression as determined by qRT-PCR.
Figure 1. Lentiviral expression vector–based shRNA clones with H1 or U6 promoter.
Figure 2. Mechanism of shRNA vector–mediated gene silencing.
The expression cassettes of all shRNA clones are fully sequenced including the promoter, sense and antisense target sequences, hairpin, termination and other linker sequences. For human kinome shRNA clones in psi-sH1 vector, we provide 1 to 3 clones for each gene, all of which have been validated to have a KD effect of 70% of the original level using the surrogate colorimetric alkaline phosphatase assay. For all other genes, lentiviral and mammalian vector-based shRNAs, we provide four shRNA clone constructs for each gene. We guarantee that at least one of the four shRNA constructs will have a knockdown effect of 70% on corresponding gene expression as determined by qRT-PCR, provided that all verification experiments are conducted in strict adherence to the protocols and procedures provided by GeneCopoeia. After adequate trouble shooting procedure is completed and it is found that none of the four shRNA constructs can achieve guaranteed level of knockdown effect, GeneCopoeia will supply a new set of 1 to 4 shRNA constructs free of charge.
GeneCopoeia offers a set of shRNA clones against 350 human kinase genes offering a guarantee of knockdown effect. Additional features include:
Figure. 3. Validation of shRNAs against human kinome members using vector-mediated (psi-sH1) shRNAs and CMV driven expression clones (in psiCheck-AP) that contain target kinase ORFs and express alkaline phosphatase (AP) for colorimetric assays. A. In the absence of shRNA, AP is expressed and its activity is quantified by colorimetric assay. B. Co-transfection of shRNA and AP-ORF expression clones, chimeric mRNA for AP-ORF is destroyed and, subsequently AP translation and its activity is reduced.
RNAi generally refers to RNA interference, which is employed by host cells to cleave double strand RNA (dsRNA) from invading microbes. Long dsRNAs can silence the expression of target genes in a variety of organisms and cell types using the RNA interference pathway. The long dsRNAs can be processed into 20-25 nucleotide small interfering RNAs (siRNAs) by an RNase III-like enzyme, Dicer. Then, the siRNAs assemble into endoribonuclease-containing complexes called RNA-induced silencing complexes (RISCs). The siRNA strands subsequently guide the RISCs to base-pair with target mRNA molecules where they then cleave and destroy the cognate RNA (Figure. 4). This is a natural mechanism whereby host organisms can defend themselves against invading microbes which use dsRNAs in their life cycles.
siRNAs were originally identified as intermediates in the RNAi pathway after induction by exogenous dsRNA as mentioned above. While the biogenesis of endogenous siRNA utilized for down-regulation of mRNA in all species is largely unknown, scientists have applied the siRNA mechanism to knock down gene expression in many experimental systems. The widely used method for gene knock down employs in vitro chemically synthesized dsRNA molecules in the size of 19-29 nucleotides with 3’ overhangs. Upon being transduced into cells, they can be similarly processed into forming RISC-shRNA complexes for subsequent gene down-regulation. Since target sequences are chosen selectively and specifically for each transcript, these chemically synthesized siRNAs become a very effective tool in down-regulating gene expression specifically in the field of reverse genetics (Figure. 4).
Figure 4. The mechanism of RNA interference (RNAi).