Yes. That’s what we are here for. As a group we have expertise in assay design, cell culture, molecular biology, bioinformatics, high-throughput and automated protocols, and more. We can also put you in touch with experts in fields such as automated image analysis. Please feel free to contact the Director or another staff member with your questions.

Here.

Anyone. We will require you to fill out an application, which is aimed at making sure you have an assay with a robust signal-to-noise and a good plan for following up on the screen. About half of screens at the DRSC are done by people from across the US or overseas and the other half by researchers local to the greater Boston area. We are here to help if you need advice, cells, protocols etc. along the way.

The Drosophila cell system has been used extensively in RNAi screening and led to new understandings relevant to diverse topics in biology and biomedicine. To read more about advantages of this system and relevance human health, please see our Fly to Human page. To search for predicted orthologs please try DIOPT.

In very brief, you develop a cell-based assay in your own lab. We can provide cherry-picks for making dsRNA for known or putative controls. We’re also happy to answer questions as you develop your assay. When you have a robust assay with working controls, then you’re ready to apply. (If you can’t quite get there without some hands-on help, contact us about an Assay Development Visit.) Once here, you are in charge of conducting your screen and making sure that you are getting meaningful results. But that’s not to say you’re on your own. We are here to help with various aspects of that process. The primary screen is performed here and then you return to your home institution for data or image analysis and follow-up tests. We continue to help through informatics support, providing collaborations on image analysis, shipping follow-up reagents, etc. even after you’ve left the DRSC. The same support is provided for off-site screens.

Announcements and breaking papers in the field can be found on our blog.

The cost of screening varies quite a bit. Library fees and other relevant information are on our costs page. Important factors affecting screen costs include (1) Are you using transfection (more costly) or bathing to deliver dsRNAs to cells? (2) Are you screening the full-genome library (more costly) or a smaller sub-library set? (3) Are you a visiting screener (unfortunately, we cannot offer housing, so you’ll have to cover that cost as well as the research costs)? And (4) Are you using a luciferase readout (tends to be more costly as compared with image-based readouts)? Feel free to get in touch for advice on how to keep costs down for your screen.

You do not have to screen at the DRSC. We will send libraries somewhere else. In the past we asked researchers to screen here and we still encourage it (makes best use of our expertise, instrumentation, etc. as well as providing a learning experience in a very exciting region in which to be doing science). However, as many researchers now have access to the necessary equipment for high-throughput screening, we no longer require that you screen on-site. We will ship any library for off-site screening, provided you have access to the appropriate assay read-out instruments for your assay, such as in your own lab or at a similar screening center. You will still have to go through the application process for full-genome screens in order to ensure that you are ready to screen before we send plates. You can expect the same level of support pre- and post-screening (i.e. advice, informatics support, access to follow-up reagents, etc.).

In general, yes. Our assay readout instruments, including the confocal screening microscope and plate-readers, have been used successfully for mammalian cells and other types of assays. We can help with some liquid handling applications as well, provided you reimburse for consumable costs and it does not interfere with reagent production and screen support, or disrupt screeners’ schedules.

Store the plate at -20. Before opening the plate seal, spin the plate down in a centrifuge. We use 2000 rpm for 2 minutes. Next, add 10 uL of ddH2O to your cherry-pick plate, bringing the total volume to 15 uL. This helps prevent loss through evaporation. You’re now ready for PCR. You can probably use as little as 0.1 uL per reaction but we typically use 1 uL per reaction. More protocol information is here.

Please see our RNAi reagent protocol page. If you still have questions, please email a DRSC staff member for help.

Information about volumes and concentrations, as well as recommended incubation times, can be found on our RNAi screening protocols page.

In general on our 384-well assay plates we provide 5 uL of dsRNA in solution at a concentration of 0.05 ug/uL for bathing and 0.016 ug/uL for transfection. The assay plates safely hold a max of about 80 uL and we recommend targeting your total volume to about 45 to 50 uL. The final concentration will depend upon your total reaction volume.

In general we recommend 1 to 5 x 10 to the 4th mL. For shorter incubations (fewer cell doubling times) you should use more cells. We recommend testing several cell concentrations as part of your assay development and optimization. The appropriate concentration differs for different screens.

We recommend adding 10 uL of cells with serum-free medium for RNAi incubation, then adding 30 uL of standard 10% FBS medium. If you want to be extra sure of the final concentration of FBS, then you can add 30 uL of 15% FBS medium instead. Media starvation for 30 minutes does not appear to have a lasting detrimental effect on cells (they recover within about a day). This answer is best understood in the context of our RNAi protocol, which can be found here.

Quality issues do sometimes arise. In some cases these reagents will be fine but in other cases there could have been mis-amplification, a rare cross-contamination event, a difference in the sequence of the reference versus the actual template, or another issue. The most cautious solution would be to re-do things from the primer stage on up. Another useful approach is to test another amplicon designed against the same gene. Please let us know about problems so we can give advice and continue to work to improve the quality of our reagent libraries.

Yes. It’s called SnapDragon. At SnapDragon, you can input fly gene IDs or a sequence. Snapdragon will help you identify primers useful to amplify an appropriate gene region (i.e. avoiding regions with sequence matches to other genes that could lead to off-target effects).

RNAi reagents are not all alike. Some do not confer robust knockdown, generating false negative results. Some have off-target effects, so they can generate false positive results. If you see that only a sub-set of reagents against a gene hit in a screen, a good first step is to check if the one(s) that hit have predicted off-targets and be suspicious when they do.

RNAi reagents with a large number of off-target effects can result in hits in many seemingly unrelated screens. These results should probably just be discarded. Our updated libraries do a better job of avoiding off-target effects but as we don’t know all of the biological ‘rules’ underlying RNAi yet, there is no perfect reagent or library. RNAi reagents that affect general growth or viability also tend to be over-represented as hits, as these affects sometimes skew results and show up as hits in various assays. Most of this type of spurious result can be avoided through careful assay design that accounts for cell number, such as by taking a ratio of an experimental readout (e.g. firefly luciferase reporter) to a ubiquitous readout (e.g. Renilla driven by a ubiquitous promoter), or by using DAPI or another method to count cells in an image-based assay.

Yes. Please see “Link to All Hits” in the Power User section of the tools overview page.

You can view full datasets associated with published screens by clicking the “Exp. Data” button for a screen on a publications page. In addition, you can search by gene name or other IDs using the Gene Lookup tool. DRSC datasets also comprise a significant percentage of data in FLIGHT and GenomeRNAi, are provided as linkouts from FlyBase gene pages, and can be searched at FlyMine as well. Our reagents and a growing number of our screen datasets are also available through NCBI’s PubChem Probe/Substance/BioAssays databases.

In general yes, we will work to make tools of particular interest to DRSC screeners or others available on our website. An example is MinoTar. Please contact the Director if you’d like to discuss an informatics collaboration of this kind.