Steps involved in RNAi screening. esiRNA, endonuclease-prepared siRNA.

Various types of RNAi chemistry. (a) Long strands of dsRNA (200–500 base pairs (bp)) are taken up by insect cells and are cleaved intracellularly into a pool of siRNA (21–27 base pairs); for mammalian cells, long strands of dsRNA are cleaved in vitro to generate endonuclease-prepared siRNA. (b) Synthetic siRNA molecules have chemically modified termini to increase stability and specificity of knockdown in cells. (c) Molecules of shRNA are ectopically expressed in cells from a U6 or H1 promoter; they can be delivered by a viral backbone with a selectable marker such as green fluorescent protein (GFP) or antibiotic resistance (for example, to puromycin (PURO)). LTR, long terminal repeat; GAG, group-associated antigen; IRES, internal ribosomal entry site. (d) The shRNA transcripts can also be expressed with additional micro-RNA (miR) sequences flanking the hairpin sequence.

Z-factor calculation and ranking of screen ‘hits’. (a) A good assay for high-throughput screening has a low s.d. and a high signal response in wells 1–96 (baseline average) relative to that in wells 97–192 (stimulated average). (b,c) In a poor assay, higher s.d. in the baseline and stimulated averages (b) or lower baseline/signal values (c) decrease the Z-factor calculated. (d) ‘Hits’ from a sample screen plate (n = 384) ranked by change in s.d. from the mean (fold): weak ‘hits’ are two to three s.d. from the mean; moderate hits are three to five s.d. from the mean; and strong hits are over five s.d. from the mean.