This choice will be dependent on the question to be addressed, the assay type and available reagents.

Traditional plate-reader screens measure emission of light from all cells in a well. This can be either luminescent read-outs (e.g. firefly or Renilla luciferase) or fluorescent read-outs (e.g. GFP). See Analyst GT.

Infrared plate-reader screens measure fluorescence (one or two channels) for in-cell westerns. Read-out data analysis can include protein-to-cell normalization for protein level quantitation. See LiCor Aerius.

High-content imaging can be used to detect the sub-cellular localization of specific proteins, organelles or DNA, such as after fluorescent labeling with antibodies or dyes. Image analysis can be used to automate or semi-automate analysis of cell shape, cell size and specific features. See Discovery-1 microscope (fluorescence, non-confocal) and Evotec Opera microscope (fluorescence, confocal).

Expert Tip: Control for Cell Number or Viability. In a screen, dsRNAs that cause cells to stop growing and/or die (such as a dsRNA directed against thread) may score as false positives or otherwise skew the data. This may be particularly relevant for plate-reader screens. You can design an internal control for cell viability or cell number to be included in your assay. For a plate-reader screen, this could be a marker (e.g. GFP, RFP) or expression construct (e.g. Renilla luciferase) that should be present in all cells that is distinct from your experimental assay marker or expression construct. For a high-content imaging screen, this could be a marker (e.g. GFP, YFP), a stain (e.g. Hoechst or DAPI), or an antibody against a protein expressed in all cells.

Please also see our Protocols page.

This choice will affect your options in terms of assays, results and protocols.

RNAi screens can be performed in

• Standard D. melanogaster cell lines (e.g. S2 cells)
• Customized D. melanogaster cell lines
• D. melanogaster primary cells

UAS-Gal4-based over-expression screens can be performed in

• Standard D. melanogaster cell lines (e.g. S2 cells)
• Customized D. melanogaster cell lines
• D. melanogaster primary cells
• Other cell types (e.g. cells from another Drosophila species)

Decisions about cell type will affect

• Your Assay (given your topic or type of assay, only certain lineages or cells with certain characteristics may be appropriate)
• Your Results (as not all cells express all genes)
• Your Protocol (most cell lines will take up dsRNA in solution but for some cell lines and for all UAS constructs, transfection is required)

Expert Tip: Bathing vs. Transfection. Many Drosophila cells will simply take up dsRNA in solution, so bathing is all that is needed to introduce the dsRNA for RNAi. However, transfection is required for some lines. Transfection is also required for introduction of DNA such as for Gal4-UAS over-expression. Thus, if you want to use one protocol for a combined RNAi and over-expression screen, you should plan to transfect.

Click to view a list of cell lines maintained at the DRSC

Please also see our Protocols page.

This choice will affect your potential results, the time it takes to perform the screen, and your protocols.

Genome-wide RNAi screens with the DRSC 2.0 library are the most commonly performed screens at the DRSC.

We also have RNAi sub-libraries (related gene sets) and a growing collection of reagents for over-expression screening. Use of a focused sub-library is faster, simply because fewer plates have to be processed to screen the full set.

Please see the Reagents page for details.

Including positive and negative controls is key. We can help with design of dsRNAs to knock down candidate control genes.

For most assays, dsRNAs (RNAi screening) or Gal4-UAS constructs (over-expression screens) targeting one or more gene that should score as a positive "hit" can be included as a positive control.

If this is not possible, another condition, perturbagen or treatment that mimics the expected result of a positive hit should be included.

Feed the Dragon. Our on-line SnapDragon software tool can be used to design dsRNA amplicons. The tool is designed to choose regions with minimal potential for off-target effects.

Take a look at what's been done here before. Before you undertake a big effort to design and carry out your screen, it is worth looking at our Completed Screens page to check what territory has already been charted in addition to searching the literature for similar or related screens done at other facilities or laboratories.

How we handle submission of similar applications. When we receive applications for very similar screens, we encourage but do not require the groups to communicate with one another. In some past cases, this has led to collaboration or co-submission of papers. But ultimately, if each screen is accepted on its own merits and both groups want to continue independently, we will not get in the way of that happening. Please see Policies for details.

Expert tip: To Screen or Not To Screen? There can be compelling reasons to move forward with a screen that is similar to a screen performed previously by you or by another group. For example, if the state-of-the-art in reagents and/or assay read-outs has changed since you or another group performed a similar screen, such that additional hits might be expected. Or if you feel the differences between you screen and another are significant enough to yield different results.

Time, money and other practical concerns will also affect your choices.

How Long Will it Take?

Before Screening. Pre-screen time factors include assay development (typically, several months or a year) and assay optimization (several weeks or months).

Screening at the DRSC. A typical genome-wide screen at the DRSC takes about 8 weeks (week 1 test plate, week 2 pilot screen, genome-wide screen weeks 3 to 8). Be aware that we schedule screeners in advance and have limited capacity. Thus, you can expect a wait-time that varies from a few weeks to a few months before you can come here to screen after it is accepted. Feel free to contact us if you are concerned about the timing of screen currently in planning or optimization stages.

After Screening. Post-screen time factors include data analysis (several weeks), screen hit validation (several weeks or months), and secondary analyses such as in vivo testing (several months or a year).

How much will it cost?

Costs. We charge a modest screener fee to partially off-set our costs. However, the most significant costs for visiting screeners involve the consumables used during screening (e.g. plastics, culture media, glow reagents), and traveling to and staying in Boston. Please see our Costs page for more details.

Options for minimizing costs. Ideally, expense alone should not prevent a good screen from happening. If cost is a concern, you can start small by trying an RNAi sub-library screen at your home institution, either prior to or instead of doing a genome-scale screen. We provide assay-ready plates (384-well format) with the dsRNAs at a reasonable fee and will try to provide advice on all aspects of screening. For on-site screens, keep in mind that different assays require different plates, reagents etc. so the assay will affect your costs. Please see the Staying in Boston page for information about minimizing the cost of your stay.

Optimization at the DRSC. If you do not have access to 384-well format equipment at your home institution, you can visit the DRSC for assay optimization (1 to 2 weeks).