"At this level of knock-on cost, the annual net saving to the public purse would be £100m, or 42% of the net saving predicted in the government's impact assessment for these areas of law.
The level of knock down in the polyclonal stable cell line was determined by Western analysis of the stable cell lines and knock down cells with at least 70% knock down were analyzed for CXCR2-mediated chemotaxis, adhesion to collgen IV and CXCR2-mediated signaling pathways.
Based on the relative expression of c-Src and c-Yes in parental cells and the level of knock-down achieved, we deduced that the residual c-Src mRNA copy number achieved with our shRNA strategy is comparable to the parental cell c-Yes mRNA copy number (Figure S1A).
The siRNA concentration corresponding to a growth inhibition of 50% was subsequently assessed for the level of knock-down induced.
At that level of knock-down, there was no detectable effect on T cell maturation (unpublished data).
Both the expression level of knock-down target proteins as well as the functional output as measured by caspase 3 activity and DNA fractionation (TUNEL) were quantified.
DOI: http://dx.doi.org/10.7554/eLife.02725.038 Finally, in order to more accurately measure the level of HR-deficiency in MMR-deficient tumors, we assessed the level of knock-down of BRCA1, BRCA2, and ATR needed to achieve an olaparib sensitivity similar to that observed in MMR-deficient cells, that is, a GI50 of 26 µM.
For these experiments, the level of gene knocked down was determined by qRT-PCR analysis.
RNAi offers the ability to target specific sequences within a gene, to target gene families and redundant genes, to target multiple members of a given pathway, and to produce graded levels of knock-downs and knock-outs.
A variety of filters can be applied to define the size of the library, e.g., selection of hairpins with known biological activity or with known levels of knock-down (quantitative PCR), and elimination of shRNAs found empirically to be "promiscuous positives" in multiple screens.
