Microarray Analysis on Irs knockout mice technology and data analysis

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Microarray Analysis on Irs knockout mice---
technology and data analysis

• Fan Li

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Outline

• Project background
• The microarray technology
• Experiment procedures
• Data storage and analysis
• Results
• Conclusions

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Irs (Insulin Receptor Substrates) Proteins in
Insulin Pathway
Insulin
Receptor
IGF1R
Irs-1
Irs-2
Cell Growth Ins Act (Muscle)
b-Cell Survival/Growth Ins Act (Liver) Brain
Growth Retinal Survival Reproduction Food Intake
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Purpose of Project

• Disruption of Irs proteins may be involved in
  type II diabetes

• We want to examine which genes are up/down
• regulated in the absent of Irs proteins

• Microarray analysis is done on Irs -/- mice and
  
• wild type mice

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Microarray Technology

• Nucleic acid probes are attached to a solid
  support, --the Chip

• Labeled cRNA target are hybridized to the
  GeneChip
• Array.

• By monitoring the amount of label associated
  with each
• probe location, presence of each kind of
  target cRNA can
• be calculated.

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A Look of the Chip
From ip.weizmann.ac.il
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Expression Overview
From www.Affymetrix.com
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Experiment Design

• Two mice with the same genotype will be studied
  individually to insure statistical significance
• Each Irs -/- mouse should be studied with a wild
  type mouse at the same time under the same
  conditions to insure comparability

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Microarray experiment on biological sample
Irs-/-
Test
tissue
WT
Control
tissue
Obtain tissue sample from mice model
mRNA extracts from tissue sample
Hybridize and wash chip
Scan chip and get data
Data sent to lab for analyze
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Good Data? Bad Data?

• Background intensity should be relatively low
• 3 region signal /5 region signal should be
  close to 1.0
• Data from the two tissue samples with the same
  experimental parameters should replicate with
  each other

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Parse and Store Microarray Data

• How to parse the data?
• ----Data are parsed by a java
  program
• automatically.
• Where to store the data?
• ---- The program inserts
  the data into the
• ArrayResult table
  in our GeneExpression
• database.

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The GeneExpression Database
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Internal Website for Data Access
User asks for specific data, request sent to
server
Server process request, query database
GeneExpression Database
Client Machine
Web Server
Database return the result set to server

• Tab delimited file saved in client machine

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The GeneSpring Software for Data Analysis
www.silicongenetics.com
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Normalization

• Normalization adjustment of individual
  hybridization intensities to balance them
  appropriately so that meaningful biological
  comparisons can be made.
• Why Normalization? unequal quantities of mRNA,
  differences in labeling or detection efficiencies
  between fluorescent markers, systematic biases,
  unequal background, noise, etc.

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Data Analysis

• Data transformation Set expression value less
  than 0.01 to 0.01
• Normalize to median divide all of the
  measurements on each chip by the median.
• Fold change of genes in experiment samples
  (Irs-/-) against control samples (WT) will be
  studied.

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Results

• The microarray analysis of hypothalamus from Irs2
  ß cells knockout mice and WT mice

Dysregulation of insulin receptor substrate 2 in
ß cells and brain causes obesity and diabetes .
J. Clin. Invest. 114908-916 (2004).
doi10.1172/JCI200422217.
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Conclusion

• Microarray analysis has helped us to identify
  some meaningful changes of gene expression level
  in Irs-/- mice models
• Computer techniques and softwares contribute to
  our microarray project, especially in the data
  storage and analysis part

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Acknowledgement

• Dr. White, Morris
• Dr. Xueying Lin
• All White Lab Members