Download Screen Guidelines

RNAi Screens at the Alberta Institute for Viral Immunology
The commercial availability of mammalian siRNA libraries combined with recent
developments in high-throughput screening technologies opens new vistas for reverse
genetic studies in complex mammalian cell culture systems. With funding provided by
the Canada Foundation for Innovation, we have established an RNAi screening core at
the Alberta Institute for Viral Immunology. The purpose of the core is to facilitate highthroughput, genome-wide RNAi screens in fly, mouse and human cell culture systems.
This guide highlights common variables that may affect the quality of raw screen data
and suggests potential solutions. This guide is only intended as a general overview of
the preparations required for an RNAi screen and we expect that each experimental
protocol will require its own unique adaptations. The process of preparing for a screen is
not a linear sequence of events and many of the steps detailed below can be performed
in parallel. This guide will not discuss issues such as data analysis and secondary
evaluation of screen hits.
1. Identify the biological question. Prior to the initiation of an RNAi screen, we
recommend precisely formulating the question you wish to address. A very generally
formulated question (e.g. which genes are required for viability of human cells in culture)
will likely yield a large and diverse set of “hits” that regulate numerous cellular events. A
more specific question (e.g. which genes modify the viability of human cells treated with
a specific toxin) will likely identify a narrower, more closely related set of genes.
2. Determine the scope of the screen. Several factors such as cost considerations,
reagent availability and user interest determine the amount of siRNAs tested in a given
screen. We are in a position to facilitate a range of screens in human, mouse and fly cell
lines that range from functional libraries (e.g. kinases/phosphatases/druggable genes)
through to whole-genome libraries. Larger screens involve greater investments in terms
of time, reagents and disposables and will likely generate a larger set of putative hits to
pursue in secondary analysis.
3. Establish a suitable assay. The success of the screen ultimately depends on the
quality of the cell culture assay used to investigate the cellular event of interest. There
are numerous plate-based assays (luminescence, fluorescence, etc.) each with their
respective merits and challenges. When selecting a cell culture assay, we recommend
considering the following variables.
Specificity. An ideal assay specifically probes the question you are interested in and
effectively discriminates against non-specific effects. In many situations, issues such as
throughput, reagent costs, instrumentation, etc. place functional limits on the assay you
can develop. In this case, a common practice is to develop a less specific primary
screen that allows high-throughput identification of all siRNAs that modify the assay
under investigation. You can then test putative hits from the primary screen in secondary
screens that are more specific for the process being investigated. For example, if you
are interested in proteins that mediate apoptosis it is possible to perform a rapid primary
screen for genes that affect general cell viability and then test candidate hits from the
primary screen in a secondary assay that specifically identifies genes that affect
apoptosis. This approach permits a cost-effective identification of a limited number of
candidate hits from a rapid screen of thousands of siRNAs. More specific secondary
assays validate the role of primary hits in the cellular pathway under investigation.
Model system. The cell type used in your screen should reliably “report” the
phenomenon you are interested in. However, practical considerations might require you
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to compromise with the ideal cell type. For example, many primary cell lines and
lymphocytes are not accessible to RNAi through standard transfection protocols. In such
cases, it is possible to perform the screen in a more assay or RNAi-friendly cell line and
test putative hits with secondary assays in a physiologically relevant cell line. This
approach allows you to identify a narrow set of potential hits in a convenient primary
assay and to verify those hits in a more relevant cell line. Given that the cell line is a
critical determinant for success of the screen, we recommend testing several cell lines
for assay sensitivity and RNAi efficiency prior to initiation of the screen. Ideally, you
would like to identify a cell type that reliably reports your assay and is accessible to
RNAi-dependent transcript destruction.
Sensitivity. Ideally, the assay will have a large signal to noise ratio, i.e. a large
difference between a positive signal and a background signal. Given, that many siRNAs
only partially knock down their targets, you can expect that many RNAi phenotypes will
correspond to partial loss-of-function phenotypes. Thus, moderate or weak dynamic
ranges reduce the likelihood of an assay successfully identifying modifier siRNAs.
Assays with large dynamic ranges will typically return the greatest number of true
positive hits and will provide greater insights into the process under investigation. It is
often instructive to test siRNAs that target known components of the pathway under
investigation prior to initiating a large-scale screen. Such siRNAs will provide an insight
into the extent to which your assay will “identify” modifiers of the process under
investigation and may ultimately serve as useful positive controls throughout the screen.
Throughput. This issue sets an effective limit on the scale of your screen. The goal
should be to screen as many samples as possible under the same experimental
conditions. Ideally, you would complete the entire screen in one run and treat and
measure each sample at the same time. In practice, you will likely have to divide your
screen into subsets that you will set up on different days, but to keep the sample-tosample variation minimal, you should aim for an assay with high throughput. This
includes establishing simple assay protocols with the possibility of automation and to
work in 96-well or 384-well plate formats. You should choose an assay that is suitable
for these plate formats. Typical readouts of high-throughput assays are fluorescent or
luminescent signals on a plate reader, or fluorescent signals on a flow cytometer. We
have several automated liquid handlers and plate readers designed to facilitate highthroughput screens and are happy to discuss the needs of an individual investigator.
Reproducibility. As screens are typically performed over a period of days to weeks,
it is valuable to eliminate assay variability as much as possible. For this reason, we
recommend that the assay chosen for the screen functions in a robust, reproducible
manner both from day-to-day and in relation to user-defined control siRNAs. To this end,
we recommend performing trial runs of several plates with control siRNAs to identify
potential sources of assay variability.
Counter detection. Variables such as cell number and cell viability can have
considerable impact on signal strength in cell culture assays. For example, cell-lethal
siRNAs will erroneously appear as modifiers of numerous biological processes and can
be a significant source of false-positives in a screen. To discriminate against such
siRNAs, we recommend developing a control assay that normalizes your assay signal to
the cell number/viability in each well. Typically, we perform RNAi screens with duplicate
measurements of the impact of each siRNA on the assay in the presence and absence
of the experimental stimulus. In this way, we can quantify and control for the direct effect
of each siRNA on the assay.
RNAi controls. The siRNA libraries are plated in such a format that each plate
contains a minimum of one column devoid of any siRNAs. We recommend spiking
control siRNAs into some of these wells as assay controls and also to facilitate plate-to-
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plate normalization. Typical control siRNAs are scrambled, non-silencing siRNAs that
serve as reliable “canaries in the coalmine” for possible non-specific effects of the
transfection procedure. Additional potential control siRNAs are pathway-specific siRNAs
that negatively or positively influence the process and assay under investigation. These
wells serve as positive controls for the detection of modifier siRNAs. We often leave
several control wells devoid of any siRNAs to confirm that the assay worked effectively
in the assay plate on the day in question. These wells serve as controls for the “health”
of the cells and the efficiency of the assay and experimental regime for each plate.
Costs. High-throughput RNAi assays are expensive. Typical costs are; plates,
transfection reagent, cell culture reagents and assay-specific reagents. We recommend
preparing a workflow of your entire screen from assay setup to secondary analysis. It is
advisable to evaluate the cost of the planned screen and make sure it matches your
budget.
4. Optimizing the RNAi transfections. Optimizing gene knock-down increases the
likelihood you will successfully identify modifiers of a particular process. When optimizing
knock-down, we recommend you consider the following variables.
Duration of RNAi incubation period. The length of time required for a given siRNA
to generate a loss-of-function phenotype can vary from gene to gene and from siRNA to
siRNA. In most screens, the investigator incubates cells with siRNAs for 48, 72, or 96
hours.
Plate format. Performing screens in 96 and 384 well formats has several
advantages. Reagent costs per siRNA are greatly reduced in comparison to larger plate
formats and the number of sample screened in a single session is greatly increased.
Thus, condensing a screen into microplate format increases throughput and decreases
reagent costs. However, multiwell plates limit the number of cells that can be screened
in a single well and are more susceptible to plate effects. Our RNAi libraries are in 96well format, and can be plated into 384-well plate if needed.
An additional option for screening is to pool the siRNAs. In this case, the investigator
pools all siRNAs targeting a single gene into a single well. Pooling has several potential
advantages and disadvantages. On the one hand, pooling decreases the amount of
reagents required for a screen and increases the throughput of a single screen. On the
other hand, it is not possible to tell how many siRNAs from a given pool generate a
particular phenotype, which may increase the number of false-positives or false
negatives. Typically, putative hits from pooled siRNA screens are evaluated by testing
each siRNA alone. If an identical phenotype is observed for more than one siRNA, the
likelihood of either the phenotype being a false-positive is reduced as it is unlikely that
two non-overlapping siRNAs will produce overlapping off-target effects.
Cell numbers. For many assays, the cell density critically influences the results.
Optimize cell numbers by plating dilution series of cells into your desired plate format
and observe their growth over the desired time frame. We recommend performing these
assays with control non-silencing siRNAs to determine the extent to which the siRNA
protocol will impact on cell growth. The cell density that should be reached by the end of
the incubation time depends on the assay that you want to perform afterwards.
siRNA concentration. Large siRNA concentrations may be toxic and cause offtarget effects and lower ones may be insufficient to cause a phenotype. Many screens
use concentrations of 10 – 30nM siRNAs, while newer generation siRNAs can be used
at concentrations as low as 5nM.
Transfection conditions. Transfection efficiency is one of the most important
variables in a screen. The better you can deplete proteins from cells, the clearer your
loss-of-function phenotypes will be. In an ideal situation, you will develop a transfection
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protocol that gives 100% transfection efficiency and causes no cellular toxicity. We
recommend testing several different transfection reagents for toxicity and knock-down
efficiency in the cell line to be used in your screen. The transfection conditions should be
optimized for the final plate format of your screen (96 or 384 well). We find functional
plate assays ideal for optimization of transfection conditions, e.g. measure loss of
GAPDH activity after incubation with GAPDH siRNA. We usually test several different
transfection reagents against each cell line and identify the transfection reagent that
gives optimal knock-down of GAPDH activity and minimal toxicity after the desired
incubation period.
5. Screen Optimization. After developing a suitable cell culture assay and determining
optimal RNAi conditions, we recommend a trial run prior to embarking on a large-scale
screen. At this stage, you should have; determined the plate format, established
protocols for all necessary instruments (e.g. liquid handlers, plate readers, etc.),
identified appropriate control siRNAs (non-silencing and positive), validated your assay
for the given plate format and confirmed detectable gene knock down in the
experimental cell type in the chosen plate format. Once these variables have been
optimized it is often instructive to perform a mini-screen of a small collection of genes.
Such screens can often be completed in a single run and will give insights into potential
areas of concern such as; plate effects, assay sensitivity, apparent hit frequency, etc.
6. Screen. When you are satisfied with your trial screen, you are in a position to
complete a larger screen. We recommend adhering to the plate layout developed during
assay optimization, including the use of non-silencing and positive control siRNAs. Most
screens involve at least duplicate measurements of each siRNA under control (e.g. no
virus/stimulus) and experimental (e.g. with virus/stimulus) assay conditions. Factors
such as handling times, reagent availability and instrument limitations determine the
number of samples you can process in a single day. The screen should be set up in
such a way that experimental and control replicates for any given plate are processed at
the same time under the same experimental conditions.
7. Recommended reading.
Reviews.
Boutros, M. and Ahringer, J., The art and design of genetic screens: RNA interference.
Nat Rev Genet 9 (7), 554 (2008).
Primary literature.
Friedman, A. and Perrimon, N., A functional RNAi screen for regulators of receptor
tyrosine kinase and ERK signalling. Nature 444 (7116), 230 (2006).
Konig, R. et al., Global analysis of host-pathogen interactions that regulate early-stage
HIV-1 replication. Cell 135 (1), 49 (2008).
Simpson, K. J. et al., Identification of genes that regulate epithelial cell migration using
an siRNA screening approach. Nat Cell Biol (2008).
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