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Microarrays: Current Capabilities and Future Directions

Microarrays: Current Capabilities and Future Directions. Jeffrey A. Lacey January 25, 2008. The Microarray Age Begins.

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Microarrays: Current Capabilities and Future Directions

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  1. Microarrays: Current Capabilities and Future Directions Jeffrey A. Lacey January 25, 2008

  2. The Microarray Age Begins SCHENA M, SHALON D, DAVIS RW, BROWN PO (1995). QUANTITATIVE MONITORING OF GENE-EXPRESSION PATTERNS WITH A COMPLEMENTARY-DNA MICROARRAY. SCIENCE 270 (5235): 467-470 OCT 20 1995.

  3. Overview • Microarray Platforms • Experimental Design • Laboratory Methods Overview • Data Analysis • Unique Applications

  4. Microarray Research Eukaryotic Research Prokaryotic Research

  5. Microarray Platforms – Spotted Arrays • Glass Slides • Corning, Erie, ArrayIt • Epoxide or aminosaline coatings • Probes robotically spotted onto slides • Cost effective for very small arrays • Cost effective for cDNA arrays • Only way to do protein microarrays

  6. Microarray Platforms – Synthesized • Agilent – Catalog and Custom • Combimatrix – Catalog and Custom • Affymetrix – Catalog and $Custom$ • Cost effective for large arrays • $100 for 15K probe array (Agilent) • $500 for 12K probes (Combimatrix) • Data quality is better than with spotted

  7. Probe Design • Probes normally between 25 and 70 nucleotides • Design from any known sequence • EST and cDNA libraries • Whole genomes/transcriptomes • Microarray probe design software • eArray (Agilent) • OligoWiz

  8. Cy 3 Labeled Sample Cy 5 Labeled Sample Experimental Design Condition A n = 3 (each) 3 Arrays 1 2 3 1 2 3 Condition B

  9. A B A B F C D C E D Experimental Design • Loop Design n = 3 (each) 6 Arrays n = 5 (each) 15 Arrays

  10. Microarray Methods Overview RNA Extraction Labeling Hybridization Imaging RT Sample A Labeled cDNA RNA + RT Sample B

  11. RNA Extraction • Kits are much better today • Extreme precautions are not needed • Check RNA quantity on Nanodrop • Check RNA quality on the Bioanalyzer • If RNA is only for microarray, additional DNAse treatments are not necessary (Ambion’s Turbo DNAse)

  12. RNA Amplification • Linear amplification (Viral promoter) • PCR based amplification • Labeling reaction incorporated in to amplification • Optimized for eukaryotic organisms • Need to add poly-A tails to prokaryotic organisms

  13. Direct Labeling of Target cDNA • Total or mRNA • cDNA (target) synthesis with fluorescent nucleotides • Template RNA Hydrolysis • Purified fluorescently labeled target

  14. Direct Labeling • First Labeling method developed • One step labeled target synthesis • Labeled nucleotides can be difficult to incorporate • Cy3/Cy5 incorporate at different rates • Creates “Dye Bias”

  15. Indirect Labeling of Target cDNA • Total or mRNA • Target cDNA Synthesis with reactive nucleotides • Template RNA hydrolysis • Addition of fluorescent dye to reactive nucleotides

  16. Indirect Labeling • Overcomes dye bias • All target synthesis is uniform • Adds bulky dye after RT • Requires additional labeling and purification steps • Incorporation rates independent of different dye properties

  17. Hybridization • Mix equal proportions of labeled target from 2 samples • Apply to surface of array in hyb chamber or under coverslip • Incubate for 4-16 hours • Wash hybridized array with series of buffers • Removes unbound target and reduces background fluorescence

  18. Image Acquisition • Scan quickly – fluorescent dyes oxidize at different rates • Perkin Elmer ScanArray HT • Will scan Agilent, Combimatrix, Spotted Arrays • Not Affymetrix • Extract data with third party software

  19. Image Analysis • If an array looks bad, leave it out

  20. Data Normalization • All data requires normalization • Dyes fluoresce differently • Adjust for spike-in controls • More probes is always better • Spatial normalization • Up-regulation ≈ Down-regulation

  21. Statistical Analysis • Free Software • R (Bioconductor, R/maanova, J/maanova) • TIGR (MIDAS) • Pay-to-play • S+ (Insightful) • GeneSpring (Agilent) • Correct p-value for large data set false discovery rate (FDR)

  22. Graphical Output

  23. Volcano Plot 12 10 8 log10(1/p-value) 6 4 2 0 -1.5 -1 -0.5 0 0.5 1 1.5 log2(intensity ratio)

  24. Gene Lists and Heat Maps

  25. Protein-Protein Interaction

  26. Cross Species Hybridization • Hybridize target from one species onto array from another species • Species must be closely related • Include as many probes as possible • Used when your organism is not sequenced • Hybridize genomic digest of your species to array to find good probes • Only include good probes in analysis

  27. Phyloarrays • Determine which species are in a mixed or environmental sample • Probes designed from variable 16s rDNA region • Template for probes is available in many large databases • Target is usually PCR amplified 16s rDNA

  28. Metabolic Function Arrays • Determine if metabolic function exists in a mixed/environmental sample • Probes on array represent all known genes of the pathway of interest • Multiple pathways can be screened on one array • Will work with RNA and genomic based hybridizations

  29. Alternative Splicing Arrays • Probes designed to represent exons and bridge introns • Applications in Eukaryotic research Probe Design Probe

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