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High-throughput gene expression

Photograph of equipment used in the Genome Technology laboratory
exploitation of genomics resources requires high-throughput monitoring of gene expression

Major investments have been made in the last few years at The James Hutton Institute and by research groups around the world to establish extensive catalogues of genes, as Expressed Sequence Tags (ESTs) and through whole or partial genome sequencing of crop plants and plant microbial pathogens. In order for these resources to be fully exploited in crop improvement and research programmes, high-throughput monitoring of gene expression is essential.

Determination of coordinated changes in gene expression over time, throughout development, or in response to manipulation, stress or pathogens, will allow novel insights into how they respond to environmental challenges. Microarray technology has been developed over the past 10 years at The James Hutton Institute by Pete Hedley and Jenny Morris and has led to the exploitation of high-throughput gene expression profiling by many collaborative groups.

State-of-the-art technology

Parallel gene expression technologies currently being developed and utilised at the James Hutton Institute include microarrays and Second Generation Sequencing (2GS), as RNA-seq. These tools enable the simultaneous analysis of the activities of many thousands of genes, providing targets for detailed downstream studies.

The primary microarray platform currently being utilised at The James Hutton Institute is the Agilent IJISS oligonucleotide array, composed of single 60mer probes representing genes from target organisms. The majority of microarrays used on-site are custom designed Agilent arrays, which allow flexible design at low cost, yet generate very high-quality expression data. These include new 60k HD microarrays for potato, barley, blackcurrant, raspberry and tomato. The group routinely processes 4-600 arrays per annum.

RNA is isolated from target tissues and QC’d using an Agilent Bioanalyzer, prior to being labeled as cDNA, and hybridised against the arrayed gene probes. The James Hutton Institute uses an Agilent Microarray G2565B scanner for data acquisition.

Heatmap imageFollowing data extraction using Agilent FE software, analysis and exploitation of data is subsequently performed using GeneSpring GX (Agilent) software, which allows identification of differentially expressed genes, comparison of expression profiles, clustering analysis and links to metabolic pathways. The Information and Computational Sciences group and BioSS are providing support for data archiving and analysis, respectively.

RNA-seq is currently being exploited for SNP marker development in barley, blackcurrant and African tree species. Library construction for Illumina RNA-seq is being optimised within the group to allow collaborative access for James Hutton Institute staff.

Collaborative microarray projects

One of the primary aims of the Genome Technology group at The James Hutton Institute is to provide optimised, low-cost access to high-throughput gene expression tools on-site through collaborative projects. Please contact Pete Hedley to discuss further.

Microarrays have been successfully applied to plant pathogens, including Pectobacterium (Ravirala et al., 2007; Coulthurst et al., 2008; Liu et al., 2008; Pritchard et al., 2009) and Erwinia amylovora (McNally et al., 2011), and also crop species, comprising potato (Ducreaux et al., 2008; Stushnoff et al., 2010; Campbell et al., 2010; Ross et al., 2011), barley (Chen et al., 2010a; Chen et al., 2010b), raspberry (Mazzitelli et al., 2007) and blackcurrant (Hedley et al., 2011). Barley arrays have also been used for cross-species analysis in wheat (Rustenholz et al., 2010) and Lolium (Byrne et al., 2010).

Studies include determination of pathogenicity factors, analysis of host-pathogen interactions, dissection of food quality traits, expression QTL (eQTL) localisation, characterisation of bud dormancy, and identification of abiotic stress responses. In addition, novel DNA-based applications have been developed, exploiting the sensitivity of Agilent arrays to enable high-throughput anchoring of physical and genetic maps in cereal species (Liu et al., 2011) and assist genome modelling in barley (Mayer et al., 2011).

Research

Areas of Interest


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The James Hutton Research Institute is the result of the merger in April 2011 of MLURI and SCRI. This merger formed a new powerhouse for research into food, land use, and climate change.