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Introduction to microarray technology

Lecture 17, Statistics 246

March 18, 2004

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Outline

A little background

Types of microarrays

cDNA arrays

Affymetrix GeneChips

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Uses and types of microarrays

Microarrays are currently used to do many different things: to detect and measure gene expression at the mRNA or protein level; to find mutations and to genotype; to (re)sequence DNA; to locate chromosomal changes (CGH = comparative genomic hybridization), and more. There are many different ways to 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 all have a

high density and number of biomolecules fixed onto a well-defined surface. Low density means 100s (e.g. protein antibodies), medium density would be 1000s to 10s of 1000s (e.g. cDNA arrays), and high-density is 100s to 1000s of 1000s, i.e.millions (e.g. short oligonucleotide arrays).

In general there are five basic aspects of microarrays: a) coupling biomolecules 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 Chain Reaction (PCR) can be helpful

This reaction is used to amplify specific DNA sequences in a complex mixture when the ends of the sequence are known. 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 excess to the denatured DNA and the temperature is lowered to 50-60˚C or even lower. The genomic DNA remains denatured, because the complementary strands are at too low a concentration to encounter each other during the period of incubation, but the specific oligonucleotides, which are at a very high concentration, hybridize with their complementary sequences in the genomic DNA.

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PCR, ctd

The hybridized oligos then serve as primers for DNA chain synthesis, which begins upon addition of a supply of dNTPs and a temperature resistant polymerase such as that from Thermus aquilus (a bacterium that lives in hot springs). This enzyme, called Taq polymerase, can extend primers at temperatures up to 72˚C. When synthesis is complete, the whole mixture is heated further (to 95˚C) to melt the newly formed duplexes. When the temperature is lowered again, a new round of synthesis takes place because excess primer is still present. Repeated cycles of synthesis (cooling) and melting (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

Hybridise target to microarray

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)

24µm24µm

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

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 probeOligonucleotide probe

**

**

*

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 Genetics Supp, Jan 1999, —two books edited by M Schena: DNA Microarrays, A Practical Approach, OUP 1999, and Microarray Biochip Technology, Eaton Publishing, 2000, —DNA Arrays or Analysis of Gene Expression by

M. Eisen and P. Brown, and —the experiences of my colleagues.

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cDNA arrays: history

cDNA microarrays have evolved from Southern blots, with clone libraries gridded out on nylon membrane filters being an important and still widely used intermediate. Things took off with the introduction of non-porous solid supports, such as glass - these permitted miniaturization - and fluorescence based detection.

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

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cDNA arrays: the process

Building the Chip:

MASSIVE PCR PCR PURIFICATION and PREPARATION

PREPARING SLIDES PRINTING

Preparing RNA:

CELL CULTURE AND HARVEST

RNA ISOLATION

cDNA PRODUCTION

Hybing the Chip:POST PROCESSING

ARRAY HYBRIDIZATION

PROBE LABELING

DATA ANALYSIS

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MASSIVE PCR PCR PURIFICATION and PREPARATION

PREPARING SLIDES

PRINTING

Building the Chip:

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

washes + 384-well format

The arrayer: high precision spotting device capable of printing 10,000 products in 14 hrs, with a plate change every 25 mins

Polylysine coating for adhering PCR products to glass slides

POST PROCESSING

Chemically converting the positive polylysine surface to prevent non-specific hybridization

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Preparing RNA:

CELL CULTURE AND 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 preferable but 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 simultaneously hybridized to chip. Hybs are performed for 5-12 hours and then chips are washed.

Two RNA samples are labelled with Cy3 or Cy5 monofunctional dyes via a chemical coupling to AA-dUTP. Samples are purified using a PCR cleanup kit.

Ratio measurements are determined via quantification of 532 nm and 635 nm emission values. Data are uploaded to the appropriate database where statistical and other 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 quality indicators, 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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Acknowledgments

UCB StatisticsUCB StatisticsCurrent and previous group Current and previous group members (you know who you members (you know who you are)are)CSIRO Image Analysis GroupMichael BuckleyMichael BuckleyRyan Lagerstorm

WEHIMany people

PMCIChuang Fong Kong

Ngai Lab (Berkeley)

Cynthia Duggan

Jonathan Scolnick

Dave Lin

Vivian Peng

Percy Luu

Elva Diaz

John Ngai

LBNL

Matt Callow

Others

Rafael Irizarry