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Microarray Analysis Susan E. Bergeson, Ph.D. Associate Professor of Pharmacology & Neuroscience

Microarray Analysis Susan E. Bergeson, Ph.D. Associate Professor of Pharmacology & Neuroscience Biotechnology Course – March 4, 2008 For PPT and practice gene lists: https ://www.ttuhsc.edu/centers/spaarc/links.aspx. Big Picture / Bottom Line:

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Microarray Analysis Susan E. Bergeson, Ph.D. Associate Professor of Pharmacology & Neuroscience

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  1. Microarray Analysis Susan E. Bergeson, Ph.D. Associate Professor of Pharmacology & Neuroscience Biotechnology Course – March 4, 2008 For PPT and practice gene lists: https://www.ttuhsc.edu/centers/spaarc/links.aspx

  2. Big Picture / Bottom Line: • Know the basics of hybridization and the tasks necessary to • yield data for the various array technologies • Annotate a gene ID list using Source • Use WebGestalt to detect pathway over-representation and • map the location of your genes onto their respective • chromosomes • -Uncover TFBS over-representation using oPOSSUM • -Be able to integrate other tasks you have already learned. • Hint: You should be able to look up information specific • to your results. • Practice, practice, practice as you will have to do all of the • tasks in a set amount of time!

  3. Have you bookmarked these web-based tools? Source: http://smd.stanford.edu/cgi-bin/source/sourceSearch -Gene ID annotation tool WebGestalt: http://bioinfo.vanderbilt.edu/webgestalt/ -For Pathway analyses and chromosomal location oPOSSUM: http://www.cisreg.ca/cgi-bin/oPOSSUM/opossum -Transcription Factor Binding Site analysis Just for fun: GeneNetwork: www.genenetwork.org - mouse genetic expression analysis Allen Brain Atlas: http://brain-map.org - in situ hybridization analysis of mouse ests

  4. How many different analyses you can do with microarrays?

  5. Microarray Analyses: • Characterization of mRNA expression • Identification of active transcription • Identification of TFBS usage • Characterization of the “histone code” • Characterization of small RNAs • Discovery of methylated DNA regions • SNP / HapMapdetection • (Genetic association studies) • 8) aCGHanalysis

  6. ? ? ?

  7. What steps can you take to produce the most quantitative mRNA results?

  8. Issues with mRNA hybridization: -mRNA represents small amount of total RNA: Purify mRNA or use oligo dT RT dye incorporation -Splice variation: Use exon specific arrays Follow up array results with exon specific RT-PCR -Gene families: cDNAs can cross hybridize due to length -Semi-quantification: Requires length and sequence independent labeling -Large amount of data: --more later!

  9. How do you perform ChIP chip?

  10. ChIP assay: 1) X-link 2) Shear DNA 3) IP with specific Ab 4) Un-link 5) RT-PCR or “chip”

  11. Significance: • Basic t-test • False Discovery Rate – FDR • ANOVA of multiple response or time course • CLUSTER analysis • PCA – principle components analysis • PLS – partial least squares • SVD – Single Value Decomposition • SAM: Significance Analysis of Microarrays (works with Excel) • http://www-stat.stanford.edu/~tibs/SAM/

  12. Just for fun! Mouse Genetics at GeneNetwork in situ info at Allen Brain Atlas

  13. Genetic Association of Alcohol Drinking with Chromosome 2 TJ Phillips et al., Mamm. Gen. (1998)

  14. Production of Recombinant Inbred Mouse Strains:

  15. Congenic 2 expression analysis: cis- and trans-regulation is apparent

  16. GeneNetwork Expression Analysis of Recombinant Inbred Strains: www.genenetwork.org Mga = MAX – gene associated Alcohol Preference: p = 0.0005

  17. GenHunter 27G = UTDDEST119241 BC055107 (Tu3a, DRR1, FAM107a) 4th http://www.brain-map.org/welcome.do

  18. See you next week! Q’s e-mail: Susan.Bergeson@TTUHSC.edu

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