A DNA expression array to detect toxic stress response in European flounder ( Platichthys flesus)

As a first stage in developing a DNA array-based approach to investigating the effects of pollutants on an environmentally relevant European fish species, we have constructed a 160-gene custom microarray for European flounder. Degenerate primers were used to amplify 110 different fragments of stress...

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Bibliographic Details
Published in:Aquatic toxicology 2003-10, Vol.65 (2), p.141-157
Main Authors: Williams, T.D., Gensberg, K., Minchin, S.D., Chipman, J.K.
Format: Article
Language:English
Subjects:
Aldehyde dehydrogenase
Animal and plant ecology
Animal, plant and microbial ecology
Animals
Applied ecology
Base Sequence
Biological and medical sciences
cDNA array
CYP1A
Cytochrome P-450 CYP1A1 - chemistry
Cytochrome P-450 CYP1A1 - genetics
DNA microarrays
Ecotoxicology, biological effects of pollution
Environmental Monitoring - methods
Female
Fish Diseases - chemically induced
Fish Diseases - genetics
Flounder
Fundamental and applied biological sciences. Psychology
Male
Marine
Microarray
Molecular Sequence Data
Oligonucleotide Array Sequence Analysis - methods
Platichthys flesus
Real-time PCR
RNA, Messenger - chemistry
RNA, Messenger - genetics
Sea water ecosystems
Sequence Analysis, DNA
Synecology
Techniques
Toxicogenomics
Water Pollutants, Chemical - poisoning
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Summary:As a first stage in developing a DNA array-based approach to investigating the effects of pollutants on an environmentally relevant European fish species, we have constructed a 160-gene custom microarray for European flounder. Degenerate primers were used to amplify 110 different fragments of stress-related and other genes from European flounder cDNA and genomic DNA. Additionally, 22 fragments were obtained by suppressive subtractive hybridisation (SSH). These fragments were cloned and sequenced, then, with additional control genes, used to create a cDNA microarray for flounder. After optimisation of the arraying process, hepatic mRNA was isolated from flounder caught in the polluted Tyne and relatively unpolluted Alde estuaries. Fluorescent cDNA probes were synthesised from the mRNA and used in dual-colour hybridisations to the microarray. A number of transcripts were differentially expressed between Tyne and Alde female flounder but these changes were not significant, due to high inter-individual variation. However, in comparisons between Tyne and Alde male flounder, 11 transcripts were found to significantly differ in expression ( P
ISSN:0166-445X
1879-1514
DOI:10.1016/S0166-445X(03)00119-X