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Lecture 1Introduction to Microarray

TechnologyStat 697K, CS 691K,

Microbio 690K

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Microarray Technology

cDNA arrays Affymetrix arrays

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Why Are Microarrays Important?

• Very large number of genes– Able to survey a large number of genes

simultaneously.

• Extremely small size– The sample to be studied can be small.

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Potential Impacts• Preventative medicine• Ability to subtype disease

– design drug therapies that treat disease causes, rather than symptoms

• Mutation/polymorphism detection (SNP’s)• Differing expression of genes over time,

between tissues, and disease states

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Application Examples• Same organism, different tissue (brain vs skin)• Same tissue, different condition (tumor vs non-tumor)• Same tissue, different organism (wild-type vs knock-out)• Time course experiments (cell cycle, development)• Different experimental conditions (glucose vs. lactose)

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Two Most Common Types of Microarrays

cDNA and Affymetrix arrays

Technology differs in:• How DNA sequences are placed on slides

– spotting vs. photolithography • photolithography: oligonucleotides are built base-by-base on

the array surface. Makes use of semiconductor technologies. • Length of DNA sequences that are placed on

slides– complete gene sequences vs. series of fragments

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cDNA Arrays• also called spotted arrays• size: standard glass slide• entire gene sequence is on slide• robotic spotting of gene sequences onto slide• from Pat Brown’s Lab at Stanford

Spotting area = 72 mm x 22 mm

Slide dimension = 76 mm x 25 mm x 1 mm (3 x 1 x .04 inches)(standard glass slide)

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Affymetrix Arrays• also called oligonucleotide arrays• size: 1.28 cm x 1.28 cm (0.5 inch x 0.5 inch)• multiple short pieces of sequence per gene on slide• photolithography to place sequences onto slide base by

base• from company Affymetrix

Source: Affymetrix Inc.

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Steps in a Microarray ExperimentBasic steps are the same for each type of array:

1) DNA complementary to genes of interest is laid out on array at defined positions

2) DNA from experimental samples is placed over the array; complementary DNA binds

3) Presence of bound DNA is detected by fluorescence following laser excitation

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Gene Expression• Each cell contains a complete set of DNA. • Only a fraction of genes are used (or

“expressed”) in any particular cell at any given time. – for example, genes specific to liver cells are not

expressed in brain cells.

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Example: Drosophila Embryo Development

Different genes are expressed at different stages of development

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• Idea: measure the amount of mRNAto see which genes are being expressedin (used by) the cell.

• Measuring proteinmight be better, but is currently harder.

Transcriptome

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Transcription

• Information contained within the DNA is transcribed into messenger RNA (mRNA) molecules.

• We study the kinds and amounts of mRNA produced by a cell to learn which genes are expressed in certain conditions.

• Gene expression is a highly complex and tightly regulated mechanism. Both an “on/off” switch and a “volume control”.

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Methods to Measure mRNA Abundance

• Northern blotting is the traditional method. Considered the gold standard, but it is done gene by gene and very time consuming– low throughput

• Expression profiling using microarray data, although noisy, can provide expression information on many thousands of genes in parallel– high throughput

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Microarrays

• DNA microarray technology relies on the hybridization properties of nucleic acids to monitor mRNA abundance on a genomic scale in different types of cells.

• “Microarrays can be used as a new kind of microscope that allows us to observe a genome’s gene expression program” ---Pat Brown.

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cDNA Microarrays

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Definitions

• Probe: DNA sequences spotted on the array, immobile substrate.

• Target: Nucleic acid samples hybridized to the array, mobile substrate.

(Some researchers use the exact opposite terminology, i.e. target is immobile, probe is mobile)

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Overview

• A robot precisely applies tiny droplets containing functional DNA to glass slides

• Researchers then attach fluorescent labels to DNA from the cell they are studying

• The labeled samples are allowed to bind to complementary DNA strands on the slides

• The slides are put into a scanning microscope that can measure the brightness of each fluorescent spot

• Brightness reveals how much of a specific DNA fragment is present, an indicator of how active it is

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Printing cDNA arrays

The arrayer is a machine that produces the cDNA glass slides

The Arrayer

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Spot size =~100 microns

Printing a cDNA array

16 print tips

100 plates = 9600 genes

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16 print-tip groups

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Hybridization

• Hybridization: the annealing of two nucleic acid strands following the base-pairing rules: – A pairs with T – C pairs with G

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Hybridization

Target Sequence

Probe Sequence

(on array)

http://www.agen.ufl.edu/~chyn 24

Hybridization

probe

target

A pairs with T

C pairs with G

www.accessexcellence.org/AB/GG/nucleic.html

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Sample Preparation• Prepare two samples for hybridization to the array:

– An experiment and control sample, i.e. tumor and normal tissue

• mRNA is extracted from samples and reverse transcribed into cDNA– makes it more stable

• During reverse transcription, a fluorescent dye is incorporated into the newly formed cDNA

• Control sample is labeled with a green fluorescent dye called Cy3

• Experiment sample is labeled with a red fluorescent dye called Cy5

• The samples are mixed and hybridized to the slide

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Scanning• The slide is scanned twice

– Once to measure red intensity– Once to measure green intensity

• The images are overlayed to produce one image(more details in next lecture on image analysis)

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Microarray Steps

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Expression Measurement • Two-dye design: measure comparative

expression level by competitive hybridization• The ratio of the red and green fluorescent

intensities for each spot is measured• The intensities indicate the relative abundance

of expression of the red and green-labeled samples, for each gene

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Measurement

M = logR/G = logR - logG• M<0: gene is over-expressed in green-labeled

sample compared to red-labeled sample. • M=0: gene is equally expressed in both

samples.• M>0: gene is over-expressed in red-labeled

sample compared to green-labeled sample.

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cDNA Microarray

• Two-dye design: measure comparative expression level by competitive hybridization

• Advantage– Relatively inexpensive – Flexibility of custom-made array

• Disadvantage– More variability

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Advantages of cDNA Arrays• Do not require full sequence information • Inexpensive hardware• Easy to design and print custom arrays

– species of interest• Simultaneous two-color hybridization• Low cost per chip• Replicate experiments are feasible

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Disadvantages of cDNA Arrays• Relatively large amount of RNA required• Cross-hybridization between spots• Chip manufacture is labor intensive• Laboratory set-up is expensive• Highly specialized equipment is required• High reagent costs (Taq, fluros)• More variability• Difficult to compare results across different

experiments

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Summary: Seven Steps

• Prepare chip using chosen DNA library• Choose cell populations

– which tissues/cells you are studying• Extract mRNA and reverse transcribe

– cDNA is more stable than mRNA• Fluorescently label cDNA’s• Hybridize to a DNA microarray• Scan the hybridized array• Interpret the scanned image

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Affymetrix Arrays

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Affymetrix Terminology• Probe: an oligonucleotide of 25 base-pairs (“25-mer”).

Each gene or portion of a gene is represented by 9 to 22 probes that uniquely identify a gene (current standard = 11).

• Perfect match (PM): A 25-mer complementary to a referencesequence of interest (e.g., part of a gene).

• Mismatch (MM): same as PM but with a single base change for the middle (13th) base. Purpose is to measure non-specific binding and background noise.

• Probe-pair: a (PM,MM) pair.

• Probe-pair set: a collection of probe-pairs for a gene.

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Each probe is width 25, or a 25-mer

11 probes for this gene

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37Source: Affymetrix Inc.

- One cell for each probe; 500,000 cells on each chip

- Millions of copies of a probe sequence in each cell

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Probe Creation and Selection• Probes are selected to have little cross-reactivity with

other genes, so that non-specific hybridization will be minimized

• Probe cells are square-shaped features on the chip containing millions of copies of a single 25-mer probe.

• The probes are synthesized in situ, using photolithography and combinatorial chemistry– similar to semiconductor technology– very fast

• Some non-specific hybridization will occur– to combat this, MM values are subtracted from PM

(more detail in later lectures)

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Sample Preparation• Only one sample is hybridized to each array • Steps in sample preparation

– Extract mRNA from cells– Use reverse transcription to turn mRNA into cDNA– Transcribe cDNA to cRNA while incorporating a biotin

label

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Hybridize, Stain, Scan• The biotin-labeled sample is hybridized to the

chip• The chip is washed and stained

– stained to help visualize the hybridization • The chip is scanned using an array scanner

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41Source: Affymetrix Inc.

Hybridization

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Hybridization

43Source: www.bioteach.ubc.ca

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Affymetrix Advantages and Disadvantages

Advantages– Very high density – Single dye label– Reproducibility

Disadvantages– Expensive

• Can’t always have replicate arrays– Limited in analysis tools– Limited number of chip types

• Chips not available for some species of interest

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Microarray Differences

One target sample per arrayTwo target samples per array

Probes are 25-mersProbes of varying length, very long

11 probe-pairs per geneOne probe per gene

cDNA arrays Affymetrix arrays

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Biological question

Experimental design

Microarray experiment

Image analysis

Expression quantification

Normalization

Estimation Testing Clustering Prediction…

Pre

-pro

cess

ing

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Gene Expression Databases

• Gene Expression Omnibus– Data repository and online resource for the storage

and retrieval of gene expression data.• Stanford Microarray Database• Gene Expression Database

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Standard Data Formats

1) Minimum Information About a Microarray Experiment - MIAME

2) MicroArray and Gene Expression - MAGE

• These provide standards for submitting and analyzing the enormous amounts of microarray data generated by different labs

• Goal: to reproduce data and exchange data between labs

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Applications of Microarrays

Common:– Measure gene expression within a single sample.– Compare gene expression in two different cell types

or tissue samples, such as in healthy and diseased tissue.

– Examine changes in gene expression over a given period of time, such as over the cell cycle.

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Applications of Microarrays

Advanced:– Infer functions of new genes based on similarities in

expression patterns with those of known genes– Understand how genes coordinate

• gene networks

– Speed up the identification of genes involved in various diseases

– Combine gene expression with other information, such as sequence, chromosome location and gene function, to help understand biological pathways

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Golub, Slonim, Lander et al. (1999) Molecular Classification of Cancer: Class Discovery and Class Prediction by Gene Expression Monitoring. Science, Vol 286, 531-537.

Molecular Classification of Cancer

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David J. Lockhart & Elizabeth A. Winzeler, 2000, Nature, VOL 405, p827

Sequence analysis of commonly regulated genes

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NGF Pathway

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Applications in Medicine

• Cancer research:– Molecular characterization of tumors on a genomic

scale (global gene expression) more reliable diagnosis and effective treatment.

• Immunology:– Study of host genomic responses to bacterial

infections

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Application Example Disease Diagnosis

• Suppose the expression patterns of various genes involved in a certain disease (such as leukemia) have been characterized in detail

• cDNA derived from suspected tissue of an individual can be hybridized to determine whether the expression patterns match that of the known disease

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Challenges• Data size is increasingly large and complex, different microarray platforms

• Incorporate very rich biological information in data analysis

• Collaborate with biologists; studies are motivated by biological questions

• Multi-disciplinary and multiple-institutional collaboration, as well as between industry and academia

• Need well-trained researchers versatile in biology, computer science and statistics

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Statistics Tutorial

• http://www.wadsworth.org/resnres/bioinfo/tut1/By Charles Lawrence and Jun S. Liu

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cDNA References• Schena M, Shalon D, Davis R, Brown P (1995) Quantitative

monitoring of gene expression patterns with a complementary DNA microarray. Science 270 467-470.

• DeRisi JL, Iyer VR, Brown PO (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278 680-686.

• Southern EM (2000) DNA microarrays. History and overview. Methods Mol Biol 170 1-15.

• Hardiman G (2002) Microarray technologies – an overview. Pharmacogenomics 3(3) 293-297.

• Nature Genetics – Chipping Forecast – devoted to microarrays (Volume 21 supplement 1-60, 1999).

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Affymetrix Reference

• Lockhart et al. (1996) Expression monitoring by hybridization to high-density oligonucleotide arrays. Nature Biotechnology 14 1675-1680.

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Credits

• Steve Qin• Jun Liu• Wing Wong• Sandrine Dudoit• Robert Gentleman• Rafael Irizarry• Yee Hwa Yang• Terry Speed• Emile F. Nuwaysir

• Rebecca Fry• Jeff Townsend• Leona Samson• Alan Robinson• Heather Harrington• Eduardo D. Sontag

These slides are based in large part on lectures by Steve Qin, University of Michigan, with generous permission.