Cell-based screening provides a powerful approach for high-throughput gene function discovery.
Screens are grouped into two types:
- Pooled format screens
- Arrayed format screens (e.g in 96- or 384-well plates
This page answers questions regarding development and optimization of arrayed cell-based assays, such as in 96- or 384-well format micro-well plates.
Arrayed pooled format screens are supported through our DRSC Drosophila cell-based RNAi reagent libraries, additional reagent libraries (e.g. miRNA-related reagents and ORFs), and state-of-the-art assay readout equipment.
With DRSC RNAi reagents, you can screen at our facility as a visiting scientist or screen at your home institution. To inquire about visiting for a screen or for an assay development visit, please see our on-site screens page.
Below we address common considerations for arrayed-format screening
What is a typical screen workflow?
- Assay development, in which you test your assay at low throughput and establish controls, such as RNAi reagents that give you the phenotype you are looking for
- Assay optimization, in which you work in the format in which you will screen (e.g. 384-well plates) and use Z prime factor analysis to test robustness
- Screening (usually broken into pilot and high-throughput phases)
- Screen data triage, analysis, and integration.
What can I screen for?
In a cell-based screen, you can monitor something as specific as changes in expression of one gene or as general as changes in cell shape. Our publications and screen summary pages give you a sense of what screens have been done by others.
What assay readout will work for my project?
This choice will be dependent on the question to be addressed, the assay type and available reagents. 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). High-content imaging can detect subcellular localization of fluorescent labeled proteins, organelles or DNA. It is also useful for cell morphology and two-color assays.
How do I avoid common pitfalls?
1. 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.
2. Avoid edge effects.
See this post for practical tips on avoiding edge effects that can be associated with micro-well plates.
What cell type should I use?
This choice will affect your options in terms of assays, results and protocols. Screens can be performed in:
Standard fly cell lines (e.g. S2, S2R+ or Kc cells)
Customized fly cell lines (e.g. CRISPR knockout cell lines or stable cell lines)
Primary cells derived from GFP-expressing embryos
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)
How do I deliver the dsRNA for a cell-based Drosophila RNAi screen?
1. “Bathing” approach
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, typically done with a lipid-based reagent, is required for introduction of DNA (such as for Gal4-UAS over-expression or of a transcriptional reporter). It can also be used to introduce the dsRNA RNAi reagent into cells for which the bathing approach does not work.
What controls do I need?
Including positive and negative controls is key.
We recommend LacZ or GFP as a negative control for RNAi.
We recommend thread (Diap1) or Rho1 as a positive control to ensure that RNAi is working in general in your cells. Thread dsRNA treatment results in robust cell death, makig it a good control for plate-reader screens. Rho1 dsRNA treatment results in large and frequently binucleate cells, making it a good control for image-based screens.
You should establish a control dsRNA that results in the phenotype of interest. We can send templates for synthesis of a few or several candidates. For most assays, dsRNAs targeting one or more gene known to be involved in the process under study can be included as a positive control. When this is not possible, another condition, perturbagen or treatment that mimics the expected result of a positive hit should be included. A caveat is that something like a drug treatment usually has a different robustness and timing as compared with RNAi.
Still have questions?
Please feel free to contact the Director for consultation at any stage, including planning your screen, obtaining funding, assay optimization, screening stages, or screen data analysis.