Name: quantitative and automated high-throughput genome-wide rnai screens in c. elegans
Uploaded: 2012-02-27T12:00:00
Duration: 10 min 58 s
Description: Watch this Scientific Journal Video about Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans at JoVE.com

We thank D. Braendle, C. L. Kurz and F. Monta&ntilde;ana-Sanchis for their contributions. This project was supported by grants from the ANR and the Conseil Regional PACA, and institutional funding from INSERM and the CNRS.

<ol>
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K., Miteva, Y., Hannenhalli, S., Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+physiological+activation+of+osmosensitive+gene+expression+mimics+transcriptional+signatures+of+pathogen+infection+in+C.+elegans.">Genetic and physiological activation of osmosensitive gene expression mimics transcriptional signatures of pathogen infection in C. elegans.</a> PLoS One. 5, e9010-e9010 (2010).</li><li>Lee, K. Z., Kniazeva, M., Han, M., Pujol, N., Ewbank, J. 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The Ewbank lab collaborates with Union Biometrica and Modul-Bio.

The current protocol allows large-scale RNAi screening in C. elegans on solid media, using the level of expression of a fluorescent reporter gene as a read-out. It has been optimized with automated tools to facilitate high-throughput assays, allowing easy and fast handling of large numbers of samples, in an efficient, reliable and reproducible manner. Other methods have been described that do not rely so heavily on automated liquid sample handling 8.
	One key organizational aspect to the protocol has been the development of a robust method of sample tracking by adapting the commercial MBioLIMS software to the RNAi screen procedure in collaboration with the company ModulBio. At every stage of the procedure, plates are now identified by barcodes generated within the LIMS. As the LIMS also records the identity of the clones in each well on each plate, going from well to putative gene is simple. And beyond this, the LIMS links to the community database WormBase, allowing direct access to molecular and functional data about any given gene. The LIMS also facilitates analyses to be conducted on the plate or even chromosome and whole-genome level. For these analyses, the data is stored in a way that is entirely compatible with previously described tools such as COPAmulti 16.
	One critical experimental point in the method is the adjustment of the quantity of food relative to the number of worms. For many assays, it is essential that worms do not starve at the end of the experiment but it is also important not to have too much food at the start. We have observed that a high density of food decreases fluorescence induction in the protocol using worms carrying the pnlp-29::GFP reporter. This may reflect a diminished infection efficiency when worms are cultured in too much bacteria (BS, unpublished results). At the same time, the culture conditions of the RNAi bacteria is also important, since this determines whether dsRNA is produced at an optimal level. Some protocols for large-scale RNAi require culture of RNAi clones on agar plates prior to seeding liquid cultures 8. We obtained robust gene silencing omitting this step. We opted for growing an excess of each culture to ensure homogenous results. The protocol described in fact provides sufficient material to conduct 2 or 3 screens in parallel. In the interest of obtaining the most robust results, we favor, however, conducting screens in duplicate using independent cultures of worms, RNAi bacteria and fungal spores.
	Another critical step is related to using medium that is neither too humid nor too dry. If the plates are not dry enough, the RNAi bacteria does not dry properly and worms will swim during the assay. This can cause problems. For example, worms are not efficiently infected by D. coniospora in liquid (C. Couillault, unpublished results). On the other hand, in dry plates, the osmolarity of the culture medium increases. This has direct effects on worm physiology 17, including the induction of some antimicrobial peptide genes, such as nlp 29 18. The volume used to dispense worms and spores is critical to allow effective and rapid drying. If more than 10 &mu;L of liquid is added to fresh plates, the wells can take many hours to dry. This can mean that it is difficult to use robotic methods to add worms to the plates. For example, each worm is dispensed by the Biosort in around 1 &mu;L of liquid. We currently distribute the worms manually as that also allows us to overcome the tendency of worms in solution to sediment.
	We previously reported an adjustment of the Biosort that allowed one 96-well plate to be analyzed in 36 minutes 19. The machine's manufacturer UBI now offers a modification of the Biosort to decrease further the analysis time. With the "FastReFlex", samples are directly routed through the flow cell for analysis, bypassing the bubble-trap filter. This results in a significant reduction in the time for data acquisition; a whole plate can be analyzed in 22 min allowing around 20 plates to be treated per day. If plates were not frozen, however, there would be an 8 hour interval between the first and last ones. Given the relatively short duration of the experiment, with the infection lasting just 18 hours, this would introduce an unacceptable variable across plates. Not only does freezing plates prior to analysis resolve this problem, but it is also essential in the context of high-throughput analyses as, with the current protocol and standard liquid-handling robots, 2 people can easily process up to 50 plates a week. Currently, we routinely screen 30 plates a week, which allows a whole-genome screen to be conducted once in less than 2 months.
	One interesting feature of this protocol is the fact that it uses solid agar, allowing assays that cannot be performed in liquid culture. As worm physiology differs depending on the culture conditions 20,21, being able to perform assays on solid medium can also complement usual liquid-based screens. For certain phenotypes (e.g. movement), the analysis can be carried out directly with 96-well solid medium plates. For the current screen, however, as the Biosort cannot sample from solid medium plates, worms are first transferred to liquid before freezing. On the other hand, the COPAS Biosort permits a multiparametric quantitative analysis compatible with the measurement of many different phenotypes. It is likely that for any given screen not all the measured parameters will be the primary read-out. Nevertheless, these measurements can be useful for several reasons. They allow one to determine whether the assay ran correctly. For example, one can check on the number and size of worms in each well. Only where an RNAi clone should block growth or reproduction should there be a deviation from the expected values. Wells with less than 20 worms or/and animals 30% smaller (TOF) compared to a standard control with no effect on growth (e.g. gfp(RNAi)) can be excluded from subsequent analyses. The function of these clones can be addressed using an alternative protocol wherein worms are exposed to the RNAi bacteria after their development is well advanced 15. The different parameters can also be used to refine candidate gene (hit) selection, for example, limiting hits only to those that affect green fluorescence but not red. The precise method chosen for selecting hits will depend on the type of screen and its exact read-out. Given the complexity of the data generated from a quantitative genome-wide screen, collaboration with a statistician may be required.
	The protocol we describe can be readily adapted to other types of assays. For example, at the infection step D. coniospora could be replaced by any other pathogen able to infect on solid media. This is potentially useful as we have found that many of the virulence factors important for Serratia marcescens infection in liquid culture do not play a role during infection on solid medium (E. Pradel, personal communication). The protocol is also fully compatible with tests of chemicals compounds 22-24, or to identify compounds with bioactivity using different libraries of bacteria 25, so should find a general utility in the C. elegans research community.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>BactoAgar </td>
 <td>BD Diagnostic Systems</td>
 <td>214010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sigma-Aldrich</td>
 <td>A9518</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BactoPeptone</td>
 <td>BD Diagnostic Systems</td>
 <td>211677</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CaCl2</td>
 <td>Any supplier</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AeraSeal adhesive film</td>
 <td>Dutscher</td>
 <td>760214</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Isopropyl &beta;-d-Thiogalactopyranoside (IPTG)</td>
 <td>Thermo Scientific</td>
 <td>AB-0481</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>K2HPO4</td>
 <td>Any supplier</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Any supplier</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>Any supplier</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Any supplier</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Na2HPO4</td>
 <td>Any supplier</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>Any supplier</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96 deep well plate </td>
 <td>Thermo Scientific</td>
 <td>AB-0932</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96 well flat plate </td>
 <td>FALCON</td>
 <td>353072</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96 well round plate </td>
 <td>FALCON</td>
 <td>353077</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tetracycline</td>
 <td>Sigma-Aldrich</td>
 <td>T8032</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Any supplier</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TECAN robot </td>
 <td>TECAN</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Liquidator96TM </td>
 <td>RAININ </td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>COPAS Biosort </td>
 <td>Union Biometrica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LIMS </td>
 <td>Modul-Bio</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

quantitative and automated high-throughput genome-wide rnai screens in c. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Watch this Scientific Journal Video about Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans at JoVE.com

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

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