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Introduction to microarray technology

Lecture 17, Statistics 246March 18, 2004

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Outline

A little backgroundTypes of microarrays

cDNA arraysAffymetrix GeneChips

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Uses and types of microarrays

Microarrays are currently used to do many different things: to detect andmeasure gene expression at the mRNA or protein level; to find mutations andto genotype; to (re)sequence DNA; to locate chromosomal changes (CGH =comparative genomic hybridization), and more. There are many different waysto do these things without microarrays, but microarrays promise a high-throughput approach to the tasks.

There are many different types of microarrays (called platforms) in use, but allhave a high density and number of biomolecules fixed onto a well-definedsurface. Low density means 100s (e.g. protein antibodies), medium densitywould be 1000s to 10s of 1000s (e.g. cDNA arrays), and high-density is 100sto 1000s of 1000s, i.e.millions (e.g. short oligonucleotide arrays).

In general there are five basic aspects of microarrays: a) couplingbiomolecules to a platform; b) preparing samples for detection; c)hybridization; d) scanning; and e) analyzing the data.

Obviously we’re interested in e), but without some knowledge of a) to d),we’d be dangerous.

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Nucleic acid hybridization: here DNA-RNA

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The rudiments of hybridization kinetics can be helpful

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Rudiments, completed

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A knolwedge of the Polymerase ChainReaction (PCR) can be helpful

This reaction is used to amplify specific DNA sequences ina complex mixture when the ends of the sequence areknown. The source is heat-denatured into single strands.Two synthetic oligonucleotides complementary to the 3’ends of the segment of interest are added in great excessto the denatured DNA and the temperature is lowered to50-60˚C or even lower. The genomic DNA remainsdenatured, because the complementary strands are at toolow a concentration to encounter each other during theperiod of incubation, but the specific oligonucleotides,which are at a very high concentration, hybridize with theircomplementary sequences in the genomic DNA.

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PCR, ctd

The hybridized oligos then serve as primers for DNAchain synthesis, which begins upon addition of asupply of dNTPs and a temperature resistantpolymerase such as that from Thermus aquilus (abacterium that lives in hot springs). This enzyme,called Taq polymerase, can extend primers attemperatures up to 72˚C. When synthesis iscomplete, the whole mixture is heated further (to95˚C) to melt the newly formed duplexes. When thetemperature is lowered again, a new round ofsynthesis takes place because excess primer is stillpresent. Repeated cycles of synthesis (cooling) andmelting (heating) quickly amplify.

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The cDNA and short (25 bp) oligo technologies in brief. Long (60-75 bp) oligo arrays are more like the cDNA ones

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cDNA clones(probes)

PCR product amplificationpurification

printing

microarray

Hybridisetarget tomicroarray

mRNA target)

excitation

laser 1laser 2

emission

scanning

analysis

0.1nl/spot

overlay images and normalise

cDNA arrays in summary

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Affymetrix GeneChips in summary(details slightly out of date)

2424__mm

Millions of copies of a specificMillions of copies of a specificoligonucleotide oligonucleotide probe probe synthesized in situ (synthesized in situ (““growngrown””))

Image of Hybridized Probe ArrayImage of Hybridized Probe Array

>200,000 different>200,000 differentcomplementary probes complementary probes

Single stranded, Single stranded, labeled RNA targetlabeled RNA target

Oligonucleotide Oligonucleotide probeprobe

* **

**

1.28cm1.28cm

GeneChipGeneChip Probe ArrayProbe ArrayHybridized Probe CellHybridized Probe Cell

Compliments of D. Gerhold

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cDNA microarrays on glass slidesA little more detail

An overview of the Brown-De Risi- Iyer technology, based on—the 2000 CSH Microarray Course notes, Nature GeneticsSupp, Jan 1999,—two books edited by M Schena: DNA Microarrays, APractical Approach, OUP 1999, and Microarray BiochipTechnology, Eaton Publishing, 2000,—DNA Arrays or Analysis of Gene Expression byM. Eisen and P. Brown, and—the experiences of my colleagues.

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cDNA arrays: history

cDNA microarrays have evolved from Southern blots, withclone libraries gridded out on nylon membrane filters beingan important and still widely used intermediate. Things tookoff with the introduction of non-porous solid supports, suchas glass - these permitted miniaturization - andfluorescence based detection.

Currently, up to about 30,000 cDNAs are spotted onto amicroscope slide.

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cDNA arrays: the processBuilding the Chip:

MASSIVE PCR PCR PURIFICATIONand PREPARATION

PREPARING SLI�DES PRINTING

Preparing RNA:

CELL CULTUREAND HARVEST

RNA ISOLATION

cDNA PRODUCTION

Hybing the Chip:POST PROCESSING

ARRAY HYBRIDIZATION

PROBE LABELING

DATA ANALYSIS

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MASSIVE PCR PCR PURIFICATIONand PREPARATION

PREPARING SLI�DES

PRINTING

Building the Chip:

Full yeast genome = 6,500 reactions IPA precipitation +�EtOH

washes + 384-well format

The arrayer: high precision spotting devicecapable of printing 10,000 products in 14 hrs,with a plate change every 25 minsPolylysine coating for adhering

PCR products to glass slides

POST PROCESSING

Chemically converting the positivepolylysine surface to prevent non-specific hybridization

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Preparing RNA:CELL CULTUREAND HARVEST

RNA ISOLATION

cDNA PRODUCTION

Designing experiments to profile conditions/perturbations/mutations and carefully controlled growth conditions

RNA yield and purity are determined by system. PolyA isolation is preferablebut total RNA is useable. Two RNA samples are hybridized/chip.

Single strand synthesis or amplification of RNA can be performed. cDNA production includes incorporation of Aminoallyl-dUTP.

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Hybing the Chip:

ARRAY HYBRIDIZATION

PROBE LABELING

DATA ANALYSIS

Cy3 and Cy5 RNA samples are simultaneouslyhybridized to chip. Hybs are performed for 5-12 hoursand then chips are washed.

Two RNA samples are labelled with Cy3 orCy5 monofunctional dyes via a chemicalcoupling to AA-dUTP. Samples are purifiedusing a PCR cleanup kit.

Ratio measurements are determined viaquantification of 532 nm and 635 nmemission values. Data are uploaded to theappropriate database where statistical andother analyses can then be performed.

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Affymetrix GeneChip expression array design

20www.affymetrix.com

21www.affymetrix.com

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Sample RNA isolation

cDNA synthesis

Biotin-labeled cRNA synthesis

cRNA fragmentation

Hybridization to array

Array wash and stain

Array scanning

Image analysis

Affymetrix processing steps Quality control procedures

Gel electrophoresis, OD

Gel electrophoresis

Examination of the intensity of the image

Examination of chip qualityindicators, and control probe sets

Gel electrophoresis, OD

Gel electrophoresis

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Cartoon version: Before labelling

Array 1 Array 2

Sample 1 Sample 2

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Before Hybridization

Array 1 Array 2

Sample 1 Sample 2

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After Hybridization

Array 1 Array 2

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Quantification

Array 1 Array 2

4 2 0 3 0 4 0 3

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Adffymetrix chip image: low res.

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AcknowledgmentsUCB StatisticsUCB StatisticsCurrent and previous groupCurrent and previous groupmembers (you know who youmembers (you know who youare)are)CSIRO Image Analysis GroupMichael BuckleyMichael BuckleyRyan Lagerstorm

WEHIMany people

PMCIChuang Fong Kong

Ngai Lab (Berkeley �) � � �Cynthia DugganJonathan ScolnickDave LinVivian PengPercy LuuElva DiazJohn Ngai

LBNLMatt Callow

OthersRafael Irizarry