Toxicogenomics: Using cDNA Microarrays to Detect Effects of Environmental Exposures Research Triangle Park, NC October 15, 2002 Mary Jane Cunningham, Ph.D. Director of Discoveries Molecular Mining Corporation 55 Rideau St. Kingston, ON Canada K7K 2Z8 613-547-9752 E-mail: [email protected]
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Toxicogenomics: Using cDNA Microarrays to Detect Effects of Environmental Exposures Research Triangle Park, NC October 15, 2002 Mary Jane Cunningham, Ph.D.
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Toxicogenomics: Using cDNA Microarrays to Detect
Effects of Environmental Exposures
Research Triangle Park, NCOctober 15, 2002
Mary Jane Cunningham, Ph.D.Director of DiscoveriesMolecular Mining Corporation55 Rideau St.Kingston, ON Canada K7K 2Z8613-547-9752E-mail: [email protected]
Definition of Toxicogenomics
The use of genomic technologies for the measurement and analysis
of gene and protein expressionin assessing the risk of
new chemical entities (NCEs)
Three Different Approaches
To study sex differences: Indirect gene expression
-Gene sequencing-Electronic comparison of cDNA library abundances
Direct gene expression
-Gene expression microarrays
Protein expression
-2D gels annotated by mass spectroscopy
Gene Sequencing
cDNA Library Technologies Oligo dT: Standard method of library production
Normalized rare clone-biased: Modified Soares* protocol used to bias library towards low abundance transcripts (75-90+% rare), increases % unique singletons in rat by 2X
Subtracted: Modified Soares* protocol used to target differentially expressed transcripts between 2 tissues, increases % unique singletons in rat by 2X
Hybridized: Top 100 highest abundance genes & 1st strand cDNA probes used to screen out high abundance clones; preferential enrichment of middle abundance clones; increases %unique singletons but only 1.5X
Summary RNA transcripts of genes known to be involved in the
response of B(a)P, APAP and CLO were observed with high abundance in rat liver cDNA libraries.
Unique genes whose functions are not yet known were also observed.
In comparisons of cDNA libraries made from both male and female rats, transcripts from both known and unknown genes were detected in higher abundance in female rats than in males.
Female Cell Line vs. Male Cell LinesHierarchical Clustering:Distance Metric - Euclidean, Complete LinkageFilter - Balanced Differential Expression >9-Fold
Results from GeneLinker TM Platinum
Summary Similar gene expression profiles from randomly-
proliferating and contact-inhibited cultures were observed for Clone 9 and H4-II-EC-3 cells.-BRL3A cells resulted in a slightly different profile.
In comparisons of expression profiles from Clone9, H4-II-EC-3, BRL3A and McA-RH7777 cells, differences were observed in the female rat-derived cell lines versus the male rat-derived cell lines. McA-RH7777 cells were observed to have the highest differences
Performed in collaboration with Oxford GlycoSciences
Summary Different gene expression profiles were observed from
liver tissues of rat treated with B(a)P, APAP and CLO. Several genes whose function is not yet known (unique
genes) were shown to be highly expressed in tissues from all 3 compounds.
A wide range of gene expression was seen in arrays from individual rat samples versus arrays using pooled samples Pooled samples tended to give an average expression
profile-an average of each individual rat’s value
Differences in gene expression profiles were also found between female and male rats. Similar differences were also detected in proteomic
profiles.
Acknowledgements cDNA Library Construction
Laura Stuve Laura KamigakiAnne Curtis Glenn Fu and group
• GEM Design, Array Manufacture and HybridizationGary Zweiger Jeff SeilhamerScott Panzer Microarray DivisionOlga Bandman
Data Analysis MethodsRoland Somogyi Stefanie FuhrmanShoudan Liang