Bioflorida Chapter Meeting Jupiter 2008v2

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bioinformatics at EcoArray, Inc.

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  • Bioflorida Chapter Meeting Jupiter 2008v2

    1. 1. Expertise in Gene Discovery, Microarray Development and Bioinformatics Heather R. Hammers BioFlorida’s Southeast Chapter Meeting Jupiter, Florida September 23, 2008
    2. 2. Business is Expanding <ul><li>Here at EcoArray, our business is expanding from environmental to all microarray applications. </li></ul><ul><li>We have been very successful with our techniques in gene discovery and proficiency with downstream applications </li></ul>
    3. 3. DNA Precision- Our Services <ul><li>cDNA library construction </li></ul><ul><ul><li>Normalized (LIB-NORM-SPE) </li></ul></ul><ul><ul><li>Non-normalized (LIB-NN-SPE) </li></ul></ul><ul><li>Suppression Subtractive Hybridization (SSH) </li></ul><ul><ul><li>Standard subtraction </li></ul></ul><ul><ul><li>Limited starting material </li></ul></ul><ul><li>Real-time PCR </li></ul><ul><li>Custom Gene Array Construction </li></ul><ul><li>Hybridizations (Array Processing) </li></ul><ul><li>Bioinformatics </li></ul>Library Construction Other Services
    4. 4. Library Construction
    5. 5. Create cDNA libraries ; Identify all expressed genes within a tissue Create subtraction libraries To identify differentially Expressed genes between two populations CLONE GENES AND SEQUENCE <ul><li>cDNA library options : </li></ul><ul><li>Non normalized </li></ul><ul><li>Normalized </li></ul><ul><li>(for removal of </li></ul><ul><li>redundant clones) </li></ul><ul><li>Subtraction library </li></ul><ul><li>options : </li></ul><ul><li>Standard subtraction </li></ul><ul><li>(500 µ gs total RNA) </li></ul><ul><li>Limited starting material </li></ul><ul><li>(1 µ g of high quality total RNA) </li></ul>
    6. 6. Custom Gene Array Construction “Microarrays”
    7. 7. You are interested in microarray technology but… <ul><li>We provide SSH libraries and normalized libraries as a first step in gene discovery </li></ul><ul><li>We can assemble your sequences, BLAST, design the probes, and manage your array </li></ul>Species of interest not yet sequenced? No commercially available array for your model organism?
    8. 8. Hybridizations (Array Processing in our Lab) Agilent Microarray Kits 1. Microarray-Based Gene Expression Analysis One-Color Quick-AMP Labeling Kit (#5190-0442) Two Color Quick-Amp Labeling Kit (#5190-0444) 2. RNA Spike-In Kits One Color (#5188-5282) Two Color (#5188-5279) 3. Gene Expression Hybridization Kits (#5188-5242) 4. Gene Expression Wash Buffers 1 and 2 5. Stabilization and Drying Solution
    9. 9. RT-PCR
    10. 10. <ul><li>Custom primers and probe sets are designed using state of the art software and design parameters. </li></ul><ul><li>Each primer set is validated for specificity and efficiency. </li></ul><ul><li>Data is analyzed ( ΔΔ Ct used to compare changes in gene expression between controls and treated). </li></ul>
    11. 11. Bioinformatics
    12. 12. Gene Sequencing and Annotation <ul><li>Sequences will be quality trimmed and vector sequences will be removed; </li></ul><ul><li>Assembly of sequences- contigs and singlets will be queried (Blast X); </li></ul><ul><li>Sequence output- excel files listing accession number, e-value, and bit score for each gene is generated and sent to users. </li></ul>“ Let DNA Precision submit your clones to our sequencing facility ”
    13. 13. Statistical Analysis Now what? We can do the analysis for you . Advanced Basic <ul><li>Filtering for outliers </li></ul><ul><li>Normalization </li></ul><ul><li>Principal component </li></ul><ul><li>ANOVA </li></ul><ul><li>Significance (p < 0.05) </li></ul><ul><li>Fold change > 2.0 </li></ul><ul><li>Gene ontology </li></ul>(optional) <ul><li>Predictive modeling </li></ul><ul><li>Biological pathways </li></ul><ul><li>Cluster Analysis </li></ul><ul><li>Biological interpretation </li></ul>
    14. 14. Expertise in Gene Discovery, Microarray Development and Bioinformatics EcoArray, Inc. www.ecoarray.com Heather R. Hammers (Bioinformatician/Scientist) [email_address]

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