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DNA microarrays for gene

expression profiling

Alberto Bernacchia

Dipartimento di Fisica E.Fermi, Università di Roma “La Sapienza”

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Motivations

We know entire DNA sequences (genomes) of different organisms.

How are the genes expressed in different conditions?

DNA microarrays may answer

this question

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DNA microarray manufacture

On each slide (few centimeters large), ≈105 spots are deposited (≈1m).

On each spot, a single gene is chemically bounded to the surface.

The entire genome in one slide

few hundred dollars each

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An experiment

Referencesample

Test sample

RNA extraction

cDNA synthesis (RT) and amplification

cRNA synthesis and labeling Cy5Cy3

Hybridization

DNA / RNA Laser Scanning

(healthy)(diseased)

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Example of scanned slide

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Example of scanned slide

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Alzheimer’s disease

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European Brain Research Institute (EBRI)Istituto di Analisi dei Sistemi ed Informatica (IASI)

AD11 transgenic mices exepress the Alzheimer’s cellular phenotype

Alzheimer’s desease

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Three brain areas: cerebral cortex, hippocampus, basal forebrain.

15 months6 months3 months

1 month

Experimental settingFour ages

Total 4x3x2=24 conditions

Two mices: anti-NGF AD11 (diseased), VHD11 (healthy)

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Reproducibility of arrays

Two conditions

healthydiseased

More conditions

diseasedhealthy

reference (wild)healthy, hyppocampus, 1 month

reference (wild)diseased, cerebral cortex, 3 months

reference (wild)healthy, basal forebrain, 6 month

reference (wild)diseased, hyppocampus, 15 months

24 conditions x 5 = 120 microarrays

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Example of scanned slide

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X

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1*backg

2* backg (50% of genes cut)

1*backg + 3 StDev(30% of genes cut)

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Log2(Red/Green)

Intensity =Red x Green

Lowess (Locally Weighted Linear Regression): data shift based on non-linear fitting

Lowess normalization

Intensity =Red x Green

Log2(Red/Green)

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Clustering samples

A distinction between two subtypes of cancer was discovered by clustering

genes

samples

clustering of samples

(columns)

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Clustering genes

temporal response of human fibroblast to serum

wound repair

genes

clustering of genes (rows)