at Cambridge University

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at Cambridge University
Analysis of gene expression in the NOD mouse
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Hi!
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Control
Chr18.A
x 3
Chr18.B

NOD
NOD.ABH(18)
Splenocyte RNA
NOD.ABH(sub18)
Sent to UK In 3 volumes ethanol _at_ 20ºC
28S
18S
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_at_Wellcome Trust, Cambridge UK
Dr Paul Lyons Laboratory
Agilent Bioanalyzer
28S
18S
Marker
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Microarray Protocol
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1st Strand cDNA synthesis
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cDNA amplification (Clontech SMART system)
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Cy3/Cy5 Incorporation - Klenow polymerase
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G-50 Sepharose column purification
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Hybridisation at 50ºC for 18 hours
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Post-hybridisation washes (SSC/SDS)
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Dry arrays, then scanned within 24 hours
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Experimental Design

• 4 experiments on Splenocyte RNA
• 2 dye reversal experiments

• 3 Pairwise comparisons within each Experiment
• Control vs C18A
• Control vs C18B
• C18A vs C18B

• Duplicated genes on each array

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The Sanger Centre
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Agilent TechnologiesG2505A Scanner
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IMAGENE 4.2
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Calculating Gene Ratios (Log2)
A
C
A
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B
B
C
Where A is NOD B is NOD.ABH(18) C is
NOD.ABH(sub18)
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Gene Ratio Agreement
Percent of Genes
Agreement between calculated and empirical gene
ratios within each experiment Where calculated
and empirical were within 25 of each other
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Identifying Candidate Genes

• Spot present in all experiments
• Greater than 2-fold change (Log21)
• Coefficient of Variation lt 20

Absolute Mean
CoeffVar
Standard Deviation
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Differentially Expressed Genes
Control vs C18A 16 genes Control vs C18B 146
genes C18A vs C18B 191 genes
From 7500 genes
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Further Investigation

• Slides from Paul Lyons, UK
• Otago Microarray Facility
• Isolation of pancreatic lymph node RNA of high
  quality

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Acknowledgements

• Paul Lyons and his research group at the MRC,
  Cambridge University, UK
• Gerry Morris and Sian Piper
• Tony Merriman, Marilyn Merriman, Peter Stockwell
  and Jade Hollis-Moffatt
• Biochemistry Department, University of Otago
• Functional Genomics, gene expression and
  proteomics Research Theme
• Marjorie MaCallum Travel Award in Biochemistry