High Throughput Screening

The High-throughput Screening (HTS) facility provides a core service for Crick research groups. It enables individual research groups to use large-scale screening technologies and approaches as part of their research arsenal.

It is staffed by highly experienced researchers who collaborate closely with Crick research groups throughout a project lifecycle from developing assays to screening through to final data interpretation and secondary screening.

The facility has particular expertise in genome-wide siRNA and RNAi screens in mammalian and Drosophila cells and has both a human siRNA Smartpool genome library and a corresponding deconvoluted library from Dharmacon, as well as a Mouse Druggable genome collection from Qiagen and a Drosophila genome RNAi library from Ambion.

In addition, the facility has assembled a collection of 4,500 well-characterised small molecule modulators (for screens where RNAi techniques are not possible), as well as smaller-scale diversity and fragment collections for use in structural studies and proof-of-concept drug development screens. Furthermore, the facility has access to arrayed collections of viable deletions of both S. cerevisiae and S. pombe that can be used for screens in these organisms.

Our equipment allows researchers to explore small to large-scale experiments in 96-, 384- or 1536-well format with many and various readouts (e.g. 2D and 3D, live cell assays, fluorescent protein and immunofluorescence studies, HTRF, luminescence etc).

Assay set up can be automated using:

  • Dual 96- and 384-well head Biomek FX liquid handling robot (Beckman)
  • Echo550 acoustic dispenser (Labcyte)
  • Preddator liquid dispenser (Redd and Whyte)7
  • BioTek ELX405 plate washers (BioTek) and Xrd384 liquid dispensers (FluidX)
  • QPixXT colony picking and replicating robot (Genetix)

Data acquisition is automated using:

  • PheraStar (BMG Labtech) and Paradigm (Molecular Devices) plate readers
  • Acumen Explorer eX3 laser scanning microplate cytometer (TTPLabtech)
  • ArrayScan VTi automated microscope (Cellomics)
  • Insight NXT automated microscope (Cellomics)
  • IncucyteFLR (Essen Bioscience)

In addition to equipment and reagents, the HTS provides all researchers with access to the accumulated data from all screens conducted in the facility. Data is held within a central bespoke database that can be interrogated and browsed enabling researchers to identify novel activities associated with their gene(s) of interest, browse the hits from screens relevant to their research and identify colleagues with whom they can collaborate.

The HTS facility is always open to collaborations outside the institute and welcomes any informal approaches.

 

Projects

The High Throughput screening facility enables research groups to access large-scale screening technologies. Primarily this takes the form of genome-wide siRNA screens although other types of screening are increasingly popular particularly screening with our bespoke collection of well-characterised small molecule modulators.

Last year we reported upgrades to our machinery aimed at increasing our capacity and throughput. Until now large-scale screening experiments have been conducted using 96- or 384-well plate formats. Even when using the 384-well format, a genome-wide siRNA screen requires 200 such plates and remains expensive. Although our improved machinery makes it possible to utilise the newer 1536-well format, there is still a question as to whether some biological responses could be compromised by this very small scale (Panel A).

This year we tested this format in a real world screening setting. We have previously conducted a genome-wide siRNA screen in 384-well format for the Developmental Signalling group aimed at identifying regulators of the TGF-b signalling pathway where the activity of the pathway can be monitored by the level of Smad2 accumulation in the nucleus (Panel B).

We repeated this screen in the 1536-well plate format to see whether we could identify any siRNA reagents that inhibited the nuclear accumulation of Smad2 in response to TGF-b in this format and if so whether hits from such a screen bore any similarity to those previously identified.

When analysed in isolation, the 1536-well screen data was internally consistent and reproducible with replicates showing good correlation with each other (Panel C). More importantly, there was a 50 per cent overlap between siRNA reagents significantly affecting Smad2 accumulation in the two formats (Panel D). This degree of overlap is similar to what we, and others, have observed when identical screens in the same format, but conducted on separate occasions, are compared.

We conclude therefore that this new format does not inherently compromise the biology under observation. Moreover, this format offers a viable approach to screening cells with limited availability e.g. stem cells, or conducting screens across many related lines at reduced cost thereby opening up whole new research possibilities.

Figure 1

Figure 1. (A). The relative sizes of an individual well from a 96-, 384- and 1536-well plate. (B). Nuclear accumulation of Smad2 in HaCat cells in response to TGF-b monitored by immunofluorescence. From these images, parameters such as cell number per well (a measure of viability) and Smad2 nuclear intensity (a measure of Smad2 accumulation) are calculated. (C). Scatter plot comparing raw ‘cell number per well’ measures for each well across two replicates from the 1536 genome screen. The correlation coefficient of 0.7 indicates a satisfactory degree of reproducibility. (D). Venn diagram indicating the overlap between siRNA reagents identified as significantly inhibiting TGF-b signalling (more than 3 s.d.) in either the 1536- or 384-well format screen

 

Michael Howell (Lead) 

michael.howell@crick.ac.uk
+44 (0) 20 7269 3170