Microarrays: B A Splitting into two single strands An - - PowerPoint PPT Presentation

1

Microarrays: An introduction to the bio- technology

Jan Komorowski

DNA – RNA – Protein transcription – translation

A B A B A B E1 E2 E3 E4 E1 E2 E3 E4 Double stranded DNA Splitting into two single strands mRNA Transcription from strand B Transcribed RNA RNA splicing results in an mRNA molecule mRNA is translated into protein

Jan Komorowski

Central dogma of molecular biology

Jan Komorowski

Gene expression patterns in Gene expression patterns in physiology and pathophysiology physiology and pathophysiology

normal resting gastric mucosa gastric cancer type A type B normal stimulated gastric mucosa

2

Jan Komorowski

1

u

5

u

7

u

8

u

6

u

2

u

4

u

3

u c f b b d e a c f

Sample Gene Expression Regulation

Jan Komorowski

Why is gene expression worth studying?

• Physiological and pathophysiological responses

are associated with specific changes in cellular gene expression

• Insight into the specific patterns of gene

expression associated with physiological and pathophysiological responses and conditions enable hypotheses about gene function and medical diagnosis

Physiology is the study of the body in a healthy state Pathophysiology is the study of disease states.

Jan Komorowski

Pharmacogenomics

• Genetic variability in drug response

– Sensitivity of some individuals to debrisoquine: certain Cytochrome P450 gene mutations incapacitate the protein enzyme and cause poor drug metabolism, eventually leading to poisoning due to the extensive exposure to the drug.

– Obtain a DNA test of the patient, make a decision based on his/her genetic make-up – Large DB’s of genetic and drug response data are commercially built for the discovery of genetically- based rules of drug selection (e.g. Genometrix Inc.)

And much more!

Jan Komorowski

Overview

3

Jan Komorowski

Hybridization

Jan Komorowski

Image after scanning

Jan Komorowski

Experiments

Extracting Data

200 10000 50.00 5.64 4800 4800 1.00 0.00 9000 300 0.03 -4.91

Genes

Cy3 Cy5 Cy5 Cy3

Cy5 Cy3

log ( )

Jan Komorowski

Hierarchical Cluster Analysis

Red > green: up-regulated Red = green Green > red : down-regulated Not available

Gene 1 Gene 7 Gene 6 Gene 8 Gene 3

Samples 1 2 3 4 5 6

Fluorescence ratios

4

Jan Komorowski

In a few words, microarrays are …

… devices for measuring

relative gene expression levels

• f a large number of pre-

selected genes

Jan Komorowski

Data analysis goals

What to study?

• Classes of experiments; changes in

expression levels in tissue samples with different e.g. diseases, treatments, environmental effects etc.

• Classes of genes; expression profiles of

genes with similar biological function

• Both of the above

Jan Komorowski

Data analysis methods

• Unsupervised learning (clustering, class

discovery); used to “discover” natural groups of genes/experiments e.g.

– discover subclasses of a form of cancer that is clinically homogenous

• Supervised learning; used to “learn” a

model of a set of predefined classes of genes/experiments e.g.

– diagnosis of cancer/subclasses of cancer

Jan Komorowski

Example

Alizadeh et al., Distinct types of diffuse large B- cell lymphoma identified by gene expression profiling, Nature, 403:503-511, 2000.

96 normal and malignant lymphocyte samples Almost 20 000 cDNA clones Two sub-clusters of DLBCL were shown to include patients with significantly different expected survival time!