- shRNAs clones are immediately available against 350 human kinome members.
- All are 19mer shRNAs designed for high KD effect and low off-target effect.
- The shRNA target sequences were cloned into a mammalian expression vector (psi-sH1). Super H1 is used to drive shRNA expression. psi-sH1 vector also contains an EGFP gene driven by CMV promoter that can be used to measure transfection efficiency.
- The KD effect of these shRNA clones in this collection have been validated through a reporter functional assay system (see principle)
For validation assays, expression clones were constructed in the psiCheck-AP vector that transcribes a chimeric mRNA. This chimeric mRNA contains both the targeted gene and a reporter gene that encodes secreted alkaline phosphatase (AP). The transcription level of targeted genes can be assayed by the enzymatic activities of AP as the result of unperturbed transcription and translation of this enzyme in the absence of shRNAs. Upon co-transduction of a shRNA clone and the corresponding psiCheck-AP expression clone, the destruction of the targeted gene mRNA by the targeting shRNA results in the degradation of the AP gene and,therefore, translation reduction of AP. The KD effect can be quantified by the colorimetric AP assay, which is illustrated in Figure 1.
Fig. 1. Validation of shRNAs against human kinome members using vector(psi-sH1) mediated 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.
The expression cassettes of all shRNA clones are fully sequenced, which includes 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 lentiviral and mammalian vector based shRNAs, we provide 4 shRNA clone constructs for each gene. We guarantee that at least one of four shRNA constructs will have a KD effect of 70% on corresponding gene expression by 70% using at least one of the 4 aforementioned validation methods, provided that all verification experiments are conducted in strict adherence to the protocols and procedures provided by GeneCopoeia. After adequate trouble shooting procedure is complete and it is found that none of the four shRNA constructs can achieve guaranteed level of KD effect, we will supply a new set of 1 to 4 shRNA constructs free of charge.
- Single gene down-regulation. The knocking down (KD) effect of the shRNA clones for a single gene can be studied and compared with that of a scrambled nucleotide control clone.
- Pathway analysis. We have grouped genes into various signal transduction, metabolic, and disease pathways and associations, as well as gene families and groups. By arraying the shRNA clones of known pathway(s) in 96 or 384 well plates, the role for a group of genes can be studied in a pathway. Currently, we offer customers with a large collection of shRNA clones against the human kinome, which can be grouped according to the functionality of each kinase.
- Also available are expression ready ORF clones for the corresponding target genes in various vector types with different reporter tags or genes. These expression ready ORF clones together with the same ORF clones that contain silent mutations in shRNA target sequence regions can be used for shRNA validation studies and gene/protein functional rescue studies for genes/proteins targeted by corresponding shRNAs.
- Knockdown Measurement:
- q-RT-PCR. Targeting of the corresponding mRNA by a shRNA expressing cassette leads to cleavage and degradation of the mRNA and subsequent reduction of protein expression. q-RT-PCR is recommended as the first step in measuring KD effect. We offer validated q-RT-PCR primers for corresponding target genes (and any other published transcripts).
- Western blot analysis. Protein down-regulation is required in most cases for gene KD studies which can be assessed by Western Blot analysis. We offer more than 2,500 monoclonal and polyclonal antibodies against human proteins for use in Western Blot analysis.
- Functional assays. KD effect of shRNAs can be studied by end-point biological and biochemical assays such as cell proliferation, colony formation, cell cycle analysis, and migration, based on the potential role played by each gene/protein.
- Functional assays by a reporter gene/protein. Alternatively, when the endogenous transcript level of gene of interest is low and/or when q-RT-PCR and Western Blot analysis is not feasible or possible, KD effect can be measured by co-transduction of shRNA clone with an expression clone plasmid which is transcribed into a chimeric mRNA transcript consisting of a reporter gene and the target gene ORF. The reporter gene encodes a reporter protein enzyme that can be used in functional assays. The destruction of the target gene mRNA by targeting shRNA results in the degradation of the reporter gene and, therefore, translation reduction of reporter protein enzyme. The KD effect can be quantified by the reporter enzymatic assays. See figure 3 for an example of this validation method.
1. Identify the gene of interest:
Please gather as much as possible of the following information: Gene Description, species, Nucleotide Accession, Entrez Gene ID, Symbols/Aliases or Catalog/product ID. You can also submit a sequence so we can search by target gene sequences homology (BLAST)
Alternatively, you can use the search tool by GeneCoppoeia - Search shRNA target by GeneCopoeia
- and order the product ID result.
2. Choose the vector(s) of interest:
Pick-up the shRNA expression system(s) you wish to use from the list (vectors details)
3. Fill out the order form:
Download the order form (order form) and fill with requested information. Send it back to us by email or FAX. We will contact you shortly to confirm: sequences, vectors and controls