Introduction to Microarrays

The Central Dogma

The Central Dogma

Hybridization

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Introduction to Microarrays

Microarrays - The Concept

Measure the level of transcript from a

very large number of genes in one go

CELL

RNA

Why not Northern?

RNA

How?

gene specific DNA probeslabeled target

gene

mRNA

Microarrays - The Technologies

Stanford Microarrays

Affymetrix

Stanford Microarrays

Glass slides

Deposition of probes

Ready-to-use array

Hybridization

Making Microarrays

1. Produce probes

2. Print by the use of a robot

• oligos• cDNA library• PCR products

Spotting - Mechanical deposition of probes

16-pin microarrayer

Sample preparation

1. Design experimentQuestion?Replicates?Test?

2. Perform experiment

4. Label RNAAmplification?Direct or indirect?Label?

wild typemutant

3. Precipitate RNAEukaryote/prokaryote?Cell wall?

mRNAmRNA

cDNAcDNA

Cy3-cDNACy5-cDNA

SAMPLE CONTROL

Stanford

microarrays

DESIGN

and ORDER

PROBES

Affymetrix GeneChip® oligonucleotide array

• 11 to 20 oligonucleotide probes for each gene

• On-chip synthesis of 25 mers

• ~20.000 genes per chip

• good quality data – low variance

Example Catalog Arrays

HumanMouseRat ArabidopsisC. elegansCanineDrosophila

E. coliP. aeruginosaPlasmodium/AnophelesVitis vinifera (Grape) Xenopus laevisYeastZebrafish

NimbleExpress™ Array Program

Fluidic Station and Scanner

The Affymetrix Genechip®

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Photolithographyin situ synthesis

Spacers bound to surface with photolabile protection groups

Mask #1Mask #2

Photolithography - Micromirrors

NimbleExpress™ Array Program

The Affymetrix GeneChip®

A gene is represented like this:

- Perfect Match (PM)- MisMatch (MM)

PMMM

PM: CGATCAATTGCACTATGTCATTTCT MM: CGATCAATTGCAGTATGTCATTTCT

The Technologies

- Costs

- Flexibility

- Data Quality

Affymetrix

Spotter

Facility setup:Stanford Microarrays < 100,000 USDAffymetrix < 250,000 USD

The Technologies – Cost

Cost pr. array Stanford Microarrays 30-50 USDAffymetrix 300-400 USD

NimbleExpress™ Array Program - a bit more expensive

The Technologies - Flexibility

Stanford microarrays: Are flexible, but

new probes must be ordered each time

Affymetrix arrays: Are not flexible, unless

you order the NimbleExpress™ chip

The Technologies - Data Quality

Reproducibility of data:(Pearson’s correlation coefficient)

Stanford microarrays: 0.80 - 0.95

Affymetrix: 0.95

The Technologies - Choice of

Stanford microarrays: If you work with unsequenced speciesLow budget

Affymetrix: Only sequenced species High data quality

Analysis of Data

Normalization:Linear or non-linear

Statistical test:student’s t-testANalysis Of VAriance

(ANOVA)Analysis:

Principle Component Analysis (PCA) Clustering and visualization

Sample PreparationHybridization

Array designProbe design

QuestionExperimental Design

Buy Chip/Array

Statistical AnalysisFit to Model (time series)

Expression IndexCalculation

Advanced Data AnalysisClustering PCA Classification Promoter Analysis Regulatory Network

ComparableGene Expression Data

Normalization

Image analysis

The DNA Array Analysis Pipeline