Breaking Report: Data Integration Tool

On my desk to read today? A paper describing a data integration tool that might be useful for analysis of RNAi screen data sets.

Renaud et al. (2012) DroPNet: a web portal for integrated analysis of Drosophila protein-protein interaction networks. Nucleic Acids Res. PubMed ID: 22641854

Breaking Report: FISH-based genome-wide screen

On my desk to read today? A new report of a screen done at the DRSC, including use of our confocal screening microscope, and using a novel fluorescence in situ hybridization (FISH)-based approach.

Joyce et al. (2012) Identification of Genes That Promote or Antagonize Somatic Homolog Pairing Using a High-Throughput FISH-Based Screen.  PLoS Genet. PubMed ID: 22589731.

Testing 1, 2, 3

Recently we have received a number of questions along the lines of, "How much dsRNA should I use and how many cells?"

If you are doing a screen with our libraries, we offer two different concentrations of dsRNA (one recommend for the bathing method, the other recommend for transfection). As a result, you can change cell number but you're stuck with our dsRNA concentrations. Thus, you should optimize things with those concentrations in mind, and for 384-well format plates.

If, however, you are designing a smaller-scale study or otherwise have control over dsRNA concentrations, you can play around with both dsRNA and cell concentrations, in various plate formats, in order to find an optimal set-up for your assay. Above is an example layout for testing what concentration of dsRNA and cells to use for an assay. In this case, we were designing a test for a 96-well format experiment using the bathing method of dsRNA delivery. We chose dsRNA directed against thread (th) as a positive control for RNAi and did a read-out of cell viability to assess knockdown. Including several negative controls (LacZ and EGFP in addition to empty wells) allowed us to make sure dsRNAs did not reach non-specific toxic levels and get a sense of the overall variability of the assay. In our test, wells in columns 1 and 2 got the smallest number of cells and less dsRNA, and wells in columns 11 and 12 got the largest number of cells and more dsRNA.

For other plate formats, other dsRNA delivery methods, different cell types, etc. you can use the same general strategy to design a test and carefully determine an optimal set of conditions for your assay. Doing this type of testing at the beginning can save time and improve results later in the process.

Breaking Report: Looking at long non-coding RNAs

On my desk to ready today? Report of an approach based on RNAi and RNA FISH that is useful for the study of long non-coding RNAs (lncRNAs).

Chakraborty (2012) Combined RNAi and localization for functionally dissecting long noncoding RNAs. Nature Methods 9:360–362.

GenomeRNAi Announces v7.0

The folks at GenomeRNAi just announced release of a version 7.0 of their database, including 28 newly available human cell-based RNAi screen data sets.

Breaking Report: Regulators of Hh Secretion

On my desk to read today? A report on a full-genome screen performed at the DRSC.

The authors include information that might be generally useful to screeners, particularly those performing or currently analyzing the results of luciferase-based assay readouts. The study also includes follow-up using in vivo RNAi.

Aikin et al. (2012) A Genome-Wide RNAi Screen Identifies Regulators of Cholesterol-Modified Hedgehog Secretion in Drosophila. PLoS One. 2012;7(3):e33665. PubMed ID: 22432040

FlyMine v33.0

FlyMine recently announced a version 33.0. Details on changes on their blog.