Table 1 Genome-wide RNAi screens in influenza virus research (2008–2013)
From: Variations in genome-wide RNAi screens: lessons from influenza research
Hao et al. (2008) [ 2 ] | Brass et al. (2009) [ 3 ] | Shapira et al. (2009) [ 4 ] | Kŏnig et al. (2010) [ 5 ] | Karlas et al. (2010) [ 6 ] | Su et al. (2013) [ 7 ] | Tran et al. (2013) [ 9 ] | |
|---|---|---|---|---|---|---|---|
No. of genes screened | 13,071 | 17,877 | 1,745 (pre-selected by yeast −2-hybrid and microarray) | 19,628 | 22,843 | 16,368 | 21,415 |
Primary screen (Source of RNAi library) | Arrayed dsRNA screen (Ambion) | Arrayed siRNA screen (Dharmacon) | Arrayed siRNA screen (Dharmacon) | Arrayed siRNA screen (Qiagen) | Arrayed siRNA screen (Qiagen) | Pooled shRNA screen (TRC) | Pooled shRNA screen (Thermo) |
Delivery method | Bathing | Transfection | Transfection | Transfection | Transfection | Lentiviral | Lentiviral |
Cell used for screen (Origin) | DL1 cells (Drosophila) | U2OS cells (Human) | HBEC cells (Human) | A549 cells (Human) | A549/293 T cells (Human) | A549 cells (Human) | A549 cells (Human) |
Virus strain | Recombinant A/WSN/33 (WSN; H1N1) virus possessing VSV-G and renilla Luciferase gene | A/Puerto Rico/8/34 (PR8; H1N1) | A/Puerto Rico/8/34 (PR8; H1N1) | Recombinant A/WSN/33 (WSN; H1N1) virus possessing renilla Luciferase gene | A/WSN/33 (WSN; H1N1) | A/WSN/33 (WSN; H1N1) | A/NY/55/2004 (NY55; H3N2) |
Detection Method & Screen parameter | Reporter (Luciferase activity) | HA expression on cell surface | Reporter (Luciferase activity) | Reporter (Luciferase activity) | NP staining/Inducible influenza-virus-specific luciferase | Deep sequencing of shRNA which pertubed the cytopahic effect by influenza virus | Deep sequencing of shRNA which pertubed the cytopahic effect by influenza virus |
Length of RNAi treament (days) | 2 days | 3 days | 3 days | 2 days | 2 days | Over 2 weeks | over 2 weeks |
Analysis time point after influenza virus infection | 24 hrs | 12 hrs | 48 hrs | 12, 24, 26 hrs | 24 hrs | 4 weeks | 72 hrs |
Methods to reduce off-target effects | Biological replicates/alternate control dsRNA | Biological replicates/ two or more siRNAs targeting to one gene | NA | Biological replicates/ two or more siRNAs targeting to one gene | Two or more siRNAs targeting to one gene | Two or more siRNAs targeting to one gene | Biological replicates |
No. of candidate genes (Primary screen) | 110 | 133 | 616 | 295 | 287 | 110 | 138 |
Steps in the viral life cycle covered by the primary screen | Uncoating, nuclear import, transcription, translation | Entry, uncoating, nuclear import, transcription, translation, HA trafficking | Entry, uncoating, nuclear import, transcription, translation, virus assembly, budding | Uncoating, nuclear import, transcription, translation | Entry, uncoating, nuclear import, transcription, translation, virus assembly, budding | Entry, uncoating, nuclear import, transcription, translation, virus assembly, budding | Entry, uncoating, nuclear import, transcription, translation, virus assembly, budding |
Secondary screen for validation | No | No | No | Wild-type A/WSN/33 (WSN; H1N1) virus | A/WSN/33 and A/Hamburg/04/2009 | High-content screening for NP staining | Viability of infected cells after siRNA transfection |
No. of candidate genes (Secondary screens) | NA | NA | NA | 219 | 168 | 38 | ~2/3 of 138 candidate genes |
Steps in the viral life cycle covered by the secondary screen | NA | NA | NA | Entry, uncoating, nuclear import, transcription, translation, virus assembly, budding | Entry, uncoating, nuclear import, transcription, translation, virus assembly, budding | Entry, uncoating, nuclear import, transcription, translation | NA |