DRSC's DIOPT and DIOPT-DIST

Sneak peak for blog readers: Our new ortholog-related tools DIOPT and DIOPT-DIST are available online and a provisional version of the paper describing the tools is also available at BMC Bioinformatics. As always, your feedback on our web-based tools is welcome!

Methods Papers

There are at least two papers in volume 782 of Methods in Molecular Biology that may be relevant to cell-based Drosophila RNAi screening:

Davidson JM, Duronio RJ. (2011) Using Drosophila S2 Cells to Measure S phase-Coupled Protein Destruction via Flow Cytometry. Methods Mol Biol. 782:205-219. PMID: 21870294.

Siudeja K, de Jong J, Sibon OC. (2011) Studying Cell Cycle Checkpoints Using Drosophila Cultured Cells. Methods Mol Biol. 782:59-73. PMID: 21870285.

Breaking Reports

On my desk to read today? Two reports related to fly RNAi studies from the Boutros group and collaborators, describing a computational approach for use with double-knockdown data and a species-spanning study related to TOR pathway signaing.

Axelsson et al. (2011) Extracting quantitative genetic interaction phenotypes from matrix combinatorial RNAi. BMC Bioinformatics. 12(1):342. PMID: 21849035.

Zacharogianni et al. (2011) ERK7 is a negative regulator of protein secretion in response to amino-acid starvation by modulating Sec16 membrane association. EMBO J. doi: 10.1038/emboj.2011.253. PMID: 21847093.

Tech Tips: Minimizing Well Volume Loss

I recently replied to a question about minimizing variability in the volume of liquid present in a well at the end of a cell-based RNAi experiment. It is important to minimize variability in the volumes well-to-well, as variability can affect the assay and/or the assay readout, causing 'noise' in the screen that obscures meaningful results. Because others might have similar concerns, I'll post a summary of my answers here.

Variability in micro-plate well volumes is usually due to equipment issues or evaporation. We recommend the following steps to limiting problems.

(1) Have your pipetting or automated liquid handling equipment calibrated, and keep it clean and clog-free. If one row or column is consistently different from the others, that can suggest you have a clog or another equipment problem.

(2) Make sure the equipment you are using is accurate in the range of volumes you are using. You would not use a P-1000 hand-held pipettor to measure 2 microliters of volume, as a P-1000 is not considered accurate in that range. Similar to this, automated liquid handling instruments are considered accurate in only within specific ranges, and should be used accordingly.

(3) In addition to using a humidified incubator, we recommend also putting the plates inside plastic containers with damp paper towels at the bottom, to help minimize evaporation loss.

(4) Take all appropriate steps to limit "false discovery" due to any cause, including: establish good positive and negative controls, make sure you have a good signal-to-noise before screening, perform replicate tests when you screen, use appropriate statistical analyses to identify screen hits, and experimentally test initial screen hits with other reagents and assays.

Breaking Reports: Validation, Screening & in vivo Technologies

On my desk to read today? A couple of new cell-based screen-related papers--one of which systematically tests orthologs of candidate genes culled from fly screens in human cells--and a report on recombinases newly available for use in Drosophila. The report on recombinases is not directly relevant to fly RNAi but I'm including it because what's described should be of general interest to Drosophila researchers--yet another new tool in our ever-growing molecular genetic toolbox.

Bai et al. (2011) Identification and characterization of a set of conserved and new regulators of cytoskeletal organisation, cell morphology and migration. BMC Biol. 9(1):54. PMID: 21834987.

Dubrovsky et al. (2011) The Drosophila FTZ-F1 nuclear receptor mediates juvenile hormone activation of E75A gene expression through an intracellular pathway. J Biol Chem. PMID: 21832074.

Nern et al. (2011) Multiple new site-specific recombinases for use in manipulating animal genomes. Proc Natl Acad Sci U S A. PMID: 21831835.

Breaking Report: Review by FAQs--Common Screening Center Questions Clarified

On my desk to read today? A review of common questions posed to screening centers--and answers to the questions--from a center in the UK. Recommended reading for potential screeners and for screening center personnel.

Jiang M et al. (2011) Tales from an academic RNAi screening facility; FAQs. Brief Funct Genomics. 10(4):227-37. PMID: 21527443

Breaking Report: a lac operon system for expression in S2 cells

On my desk to read today? A report describing use of the lac operon system for inducible expression of a transgene in S2 cells.

Wakiyama et al. (2011) Inducible protein expression in Drosophila Schneider 2 cells using the lac operator-repressor system. Biotechnol. Lett. PMID: 21826399.

Batch Search of TRiP Tools

Sneak peek for blog readers: here's a link to a batch search tool for TRiP stocks. Feedback welcome. It will be linked to from TRiP web pages on in vivo fly RNAi resources soon. Upload files should be .txt with one gene symbol (or other identifier) per line.

The DKFZ's GenomeRNAi turns 5.0

I just learned that the Boutros group at DKFZ has launched a "5.0" version of their GenomeRNAi database. Results from DRSC screens, other fly and mammalian cell-based screens, and fly in vivo screens are searchable at GenomeRNAi. Check it out!

Publications on GenomeRNAi are listed here.

Cross-Species Validation with Live Imaging--Cell Cycle Screen Hits

Also on my desk today? A report from Sironi et al. using living mammalian cell imaging and analysis to follow up on hits from RNAi screens related to the cell cycle performed in other species.

Sironi et al. (2011) Automatic quantification of microtubule dynamics enables RNAi-screening of new mitotic spindle regulators. Cytoskeleton 68:266-278.

New Editorial & Report: Phagocytosis of Bacteria

On my desk this morning? A review by Flannagan and Grinstein on the use of knockdown approaches in Drosophila and zebrafish to study phagocytosis of bacteria. The original article is Ulvila et al. (2011). Info on both below.

Flannagan & Grinsten (2011) Fly fishing with RNAi catches novel effectors of phagocytosis. J. Leukoc. Biol. 89:643-645.

Ulvila et al. (2011) Cofilin regulator 14-3-3[] is an evolutionarily conserved protein required for phagocytosis and microbial resistance. J. Leukoc. Biol. 89:649-659.