Publication Abstract

Construction of subtracted EST and normalised cDNA libraries from liver of chemical-exposed three-spined stickleback (Gasterosteus aculatus) containing pollutant-responsive genes as a resource for transcriptome analysis

of subtracted EST and normalised cDNA libraries from liver of chemical-exposed three-spined stickleback (Gasterosteus aculatus) containing pollutant responsive genes as a resource for transcriptome analysis

M.M. Brown, T.D. Williams, J.K. Chipman, I. Katsiadaki, M. Sanders and J.A. Craft

The three-spined stickleback (Gasterosteus aculeatus) with its small size and short life cycle is ideally suited to laboratory studies, while its wide distribution in the northern hemisphere gives it great potential as a sentinel organism. In the setting of a UK-wide collaboration (Fish Toxicogenomics) an expression microarray is under development for transcriptome analysis of chemical responses in populations of G. aculeatus under lab and field conditions. Although several EST libraries are currently available for G. aculeatus none are from chemical-exposed fish and thus are unlikely to include a full set of pollutant-responsive genes. To harvest such transcripts from G. aculeatus, cDNA libraries were produced from liver of chemical-exposed (copper, ethynyl oestradiol, di-benz(a,h)anthracene) mature males. Pooled polyA+ RNA (chemical-exposed/control for each time-point/concentration) was used to generate two normalised full-length cDNA libraries by different methods; 1) partial subtraction of polyA+ RNA against solid-phase cDNA using magnetic bead technology and 2) degradation of double stranded cDNA formed by abundant transcripts. To enrich for pollutant-responsive genes a subtracted EST library was also generated using suppression subtractive hybridisation (pooled chemical-exposed minus control transcripts). For each library ~1.5K clones were sequenced and characterised using Blast2GO. All libraries contained pollutant-responsive transcripts not previously available while additionally the subtracted library was generally enriched ~1.2 -10 fold for transcripts expected to be induced in response to the pollutants.

Reference

M.M. Brown, T.D. Williams, J.K. Chipman, I. Katsiadaki, M. Sanders and J.A. Craft (2008) Construction of subtracted EST and normalised cDNA libraries from liver of chemically-exposed three-spined stickleback (Gasterosteus aculatus) containing pollutant responsive genes as a resource for transcriptome analysis. Marine Environmental Research 66:127-130

M.M. Brown, T.D. Williams, J.K. Chipman, I. Katsiadaki*, M. Sanders* and J.A. Craft

Marine Environmental Research 66, 127-130