Table 1.
Comparison of JAK/STAT RNAi screens
| Experimental condition | Baeg | Müller |
|---|---|---|
| Biological differences | ||
| Pathway stimulation | Endogenous Upd2 | Ectopically expressed Upd-GFP |
| Cell line | S2-NP | Kc167 |
| Screening procedure | ||
| Coverage of library | 21,300 dsRNAs | 20,026 dsRNAs |
| Screening reporter | 10xStat92E-luciferase (FL) containing five tandem repeats of a 441bp fragment from the socs36E enhancer (each with four potential Stat92E binding sites) | p6x2xDrafLuc containing six repeats of a 165 bp fragment from the raf promoter (each with two Stat92E binding sites) |
| Co-reporter | pAct-RL | pAct-RL |
| RNA concentration per well | 80 ng/well | 500 ng/well |
| Cells seeded per well of a 384-well plate | 40,000 | 15,000 |
| Transfection of reporter | Per well | In batch |
| dsRNA uptake | Transfection | Bathing + SID-1 dsRNA trans-porter |
| Time for RNAi | 4 days | 5 days |
| Replicate datasets | Two | Two |
| Data processing | ||
| Data normalisation | Fold SD from the plate mean of FL/RL ratio for each plate | Fold MAD from the plate median of FL channel for each plate |
| Selection of positive regulators | <2 SD below plate mean | <2 MAD below plate median |
| Selection of negative regulators | >3 SD above plate mean | >2 MAD above plate median |
| Exclusion of genes | Genes not annotated by BDGP, ribosomal proteins, proteins involved in RNA processing and translation | Previously published cell viability modifiers, treatments with high variability between replicates, treatments with z-scores >2 or <2 in the RL channel, genes with phenotypes in other screens |
| False-positive rate (determined by re-screens of primary hits) | 29% | 15% |
| Human homologs of hits | 73% | 74% |