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Keynote Topics: 1. Human genome annotations: emergence of non-coding transcripts

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Affymetrix User’s Group Meeting Boston, MA May 2005. Keynote Topics: 1. Human genome annotations: emergence of non-coding transcripts

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slide1

Affymetrix User’s Group Meeting Boston, MA May 2005

  • Keynote Topics:
  • 1. Human genome annotations: emergence of non-coding transcripts
  • -tiling arrays: study gene regulation (complement expression arrays),
  • explore genome functions including: DNA methylation, origins of replication, mapping sites of TF binding
  • 2. mRNA transcript variation across ‘druggable’ genome: novel targets from exon array data
  • - exon arrays: study alternative splicing, explore transcript based pathways vs gene based pathways
  • 3. Enabling microarrays for routine clinical use
  • personalized medicine
  • -example = SNP chips (mapping & linkage analysis)
  • -10K, 100K & 500K SNP chips
  • - Serono Identifies 80 genes involved in MS using 100K SNPs
slide2

Data Analysis Track:

  • 1. Microarray Expts: Conceptual view & Practical Considerations
  • - Experiment design-begin w/end in mind, focus on questions seeking to be addressed & have specific goals (i.e. transcriptional changes vs cluster analysis)
  • - Replicates: biological vs technical, no need for technical replicates if sample preparation, etc is done consistently, # of biological replicates depends on focus of experiment, pooling – suppresses changes
  • - data analysis: 1) look at the data statistically 2) look at the data biologically
  • 2. Plier algorithm: higher differential sensitivity for low expressors
  • - signal detection algorithm
  • - normalization
  • - background correction (PM-MM)
  • 3. Statistical tools & data visualization
  • - RMA = robust multi-chip average
  • T-test (ANOVA) – compare signal across biological replicates
  • software
slide3

Software

  • Affy Array Assist Lite (Stratagene)
  • – download for free off Affymetrix website (www.affymetrix.com)
  • Poster
  • Effects of RNA Degradation on Gene Expression Analysis of Human Post-mortem Tissues
  • – Jerry Lee, Dept of Molecular Medicine, Neurocrine Biosciences, Inc., San Diego, CA
  • Summary: RNA from Human Brain (and other tissues) & RNA from Rat duodenum was used to address how RNA quality affects gene expression data generated using Affy chips
  • – Conclusion: Degraded RNA gave comparable signal to “good quality” RNA
slide4
Effects of Exercise on Gene Expression in Bone & Brain Tissues from different strains and sexes of mice

Updates:

1. Exercise Program (sufficient to produce significant changes in skeletal system)

-mice ~6 months old (growth should have stopped)

-mice randomly selected to exercise

-10 degree incline (treadmill design)

-exercise during dark cycle (when mice naturally more active)

-30 min/day, 5 days/wk, 6 wks total

2. Experimental Considerations

a. RNA quality

b. # of replicates/pooling

c. chip array type? ($175 price difference)

1. 430A 2.0 - $350/chip, 14,000 genes

2. 430 2.0 - $525/chip, 39,000 transcripts

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