DNA MicroarraysDNA Microarrays

M. Ahmad Chaudhry, Ph. D.M. Ahmad Chaudhry, Ph. D.

Director Microarray FacilityDirector Microarray Facility

University of VermontUniversity of Vermont

Outline of the lectureOutline of the lecture

• Overview of Micoarray Technology• Types of Microarrays• Manufacturing

• Instrumentation and Softwares• Data analysis

• Applications

• Mainly used in gene discovery

Microarray DevelopmentMicroarray Development

• Widely adopted

• Relatively young technology

Evolution & IndustrializationEvolution & Industrialization

• 1994- First cDNAs arrays were developed at Stanford University.

• 1996- Commercialization of arrays

• 1997-Genome-wide Expression Monitoring in S. cerevisiae

• Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of genes (usually between 500-20,000)

• Via a conventional DNA hybridization process, the level of expression/activity of genes is measured

• Data are read using laser-activated fluorescence readers

• The process is “ultra-high throughput”

What are Microarrays?What are Microarrays?

Why use Microarrays?Why use Microarrays?• What genes are Present/Absent in a cell?

• What genes are Present/Absent in the experiment vs. control?

• Which genes have increased/decreased expression in experiment vs. control?

• Which genes have biological significance?

Why analyze so many genes?Why analyze so many genes?

• Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function.

• Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways

The 6 steps of a DNA microarray The 6 steps of a DNA microarray experiment (1-3)experiment (1-3)

1. Manufacturing of the microarray

2. Experimental design and choice of reference: what to compare to what?

3. Target preparation (labeling) and hybridization

The 6 steps of a microarray experiment (4-6)The 6 steps of a microarray experiment (4-6)

4. Image acquisition (scanning) and quantification (signal intensity to numbers)

5. Database building, filtering and normalization

6. Statistical analysis and data mining

GENE EXPRESSION ANALYSIS WITH MICROARRAYS

DNA Chips

Miniaturized, high density arrays of oligos (Affymetrix Inc.)

Printed cDNA or Oligonucleotide Arrays Robotically spotted cDNAs or Oligonucleotides • Printed on Nylon, Plastic or Glass surface

Affymetrix MicroarraysAffymetrix Microarrays

Involves Fluorescently tagged cRNA • One chip per sample• One for control• One for each experiment

Glass Slide Microarrays Involves two dyes/one chip

• Red dye• Green dye• Control and experiment on same chip

Gene Chip Technology Affymetrix Inc

Miniaturized, high density arrays of oligos 1.28-cm by 1.28-cm (409,000 oligos)

Manufacturing Process

Solid-phase chemical synthesis and Photolithographic fabrication techniques employed in semiconductor industry

Selection of Expression ProbesSelection of Expression ProbesSet of oligos to be synthesized is defined, based on its ability to Set of oligos to be synthesized is defined, based on its ability to hybridize to the target genes of interesthybridize to the target genes of interest

Probes

Sequence

Perfect Match

MismatchChip

5’ 3’

Computer algorithms are used to design photolithographic masks for use in manufacturing

• Each gene is represented on the probe array by multiple probe pairs

• Each probe pair consists of a perfect match and a mismatch oligonucleotide

Photolithographic SynthesisPhotolithographic Synthesis

Manufacturing ProcessManufacturing ProcessProbe arrays are manufactured by light-directed chemical Probe arrays are manufactured by light-directed chemical synthesis process which enables the synthesis of hundreds of synthesis process which enables the synthesis of hundreds of thousands of discrete compounds in precise locationsthousands of discrete compounds in precise locations

Lamp

Mask Chip

Affymetrix Wafer and Chip FormatAffymetrix Wafer and Chip Format

1.28cm

20 - 50 µm

20 - 50 µm

Millions of identical oligonucleotide

probes per feature

49 - 400 chips/wafer

up to ~ 400,000 features/chip

Creating TargetsCreating Targets

Reverse Transcriptase

in vitro transcription

mRNA

cDNA

Target

cRNA

RNA-DNA HybridizationRNA-DNA Hybridization

probe setsDNA

(25 base oligonucleotides of known sequence)

TargetsRNA

Non-Hybridized Targets are Washed AwayNon-Hybridized Targets are Washed Away

“probe sets” (oligo’s)

Targets(fluorescently tagged)

Non-bound ones are washed away

Target PreparationTarget Preparation

cDNA

Wash & Stain

Scan

Hybridize

(16 hours)

mRNAAAAA

B B B B

Biotin-labeled transcripts Fragment

(heat, Mg2+)

Fragmented cRNA

B B

B

B

IVT(Biotin-UTPBiotin-CTP)

GeneChipGeneChip®® Expression Analysis Expression Analysis

Hybridization and StainingHybridization and Staining

Array

cRNA Target

Hybridized Array

Streptravidin-phycoerythrinconjugate

InstrumentationAffymetrix GeneChip System

3000-7G Scanner450 Fluidic Station

640 Hybridization Oven

Currently Available GeneChips

B. subtilis

Barley Genome Array

Bovine Genome Array

C. elegans Genome Array

Canine Genome Array

Chicken Genome Array

Drosophila Genome Arrays

E. coli Genome Arrays

Human Genome Arrays

Maize Genome Array

Mouse Genome Arrays

P. aeruginosa Genome Array

Plasmodium/Anopheles Genome Array

Porcine Genome Array

Rat Genome Arrays

Rice Genome Array

Soybean Genome Array

Sugar Cane Genome Array

Vitis vinifera (Grape) Array

Wheat Genome Array

Xenopus laevis Genome Array

Yeast Genome Arrays

Zebrafish Genome Array

Arabidopsis Genome Arrays

Custom GeneChips

Affymetrix offers over 120 prokaryotic arrays that are manufactured by Nimblegen Inc.

Custom GeneChips are also available for both Eukaryotic and Prokaryotic systems.

Quality Control IssuesQuality Control Issues

• RNA purity and integrity• cDNA synthesis efficiency• Efficient cRNA synthesis, labeling and

fragmentation• Target evaluation with Test Chips

GENE EXPRESSION ANALYSIS WITH MICROARRAYS

DNA Chips

Miniaturized, high density arrays of oligos (Affymetrix Inc.)

Printed cDNA or Oligonucleotide Arrays Robotically spotted cDNAs or Oligonucleotides • Printed on Nylon, Plastic or Glass surface

Microarray of thousands of genes on a glass slide

Spotted arraysSpotted arrays

1 nanolitre spots90-120 um diameter

384 well source plate

chemically modified slides

steel

spotting pin

TheThe processprocessBuilding the chip:

MASSIVE PCR PCR PURIFICATION and PREPARATION

PREPARING SLIDES PRINTING

RNA preparation:

CELL CULTURE AND HARVEST

RNA ISOLATION

cDNA PRODUCTION

Hybing the chip: POST PROCESSING

ARRAY HYBRIDIZATION

PROBE LABELING

DATA ANALYSIS

Building the chipBuilding the chip

Arrayed Library(96 or 384-well plates of bacterial glycerol stocks)

PCR amplificationDirectly from colonies withSP6-T7 primers in 96-well plates

Consolidate into 384-well plates

Spot as microarrayon glass slides

Sample preparationSample preparation

HybridizationHybridizationBinding of cDNA target samples to cDNA probes on the slideBinding of cDNA target samples to cDNA probes on the slide

cover

slip

Hybridize for

5-12 hours

LABEL

3XSSC

HYB CHAMBER

ARRAY

SLIDE

LIFTERSLIP

SLIDE LABEL

• Humidity• Temperature• Formamide

(Lowers the Tm)

Hybridization chamberHybridization chamber

Expression profiling with DNA microarraysExpression profiling with DNA microarrays

cDNA “A”Cy5 labeled

cDNA “B”Cy3 labeled

Hybridization Scanning

Laser 1 Laser 2

+

Analysis Image Capture

Image analysisImage analysis

• The raw data from a cDNA microarray experiment consist of pairs of image files, 16-bit TIFFs, one for each of the dyes.

• Image analysis is required to extract measures of the red and green fluorescence intensities for each spot on the array.

Image analysisImage analysis

GenePix

Image analysisImage analysis

1. Addressing. Estimate location of spot centers.

2. Segmentation. Classify pixels as foreground (signal) or background.

3. Information extraction. For each spot on the array and each dye

• signal intensities;• background intensities; • quality measures.

R and G for each spot on the array.

Biological Question

Sample PreparationMicroarray

Life Cycle

Data Analysis & Modelling

Microarray Reaction

MicroarrayDetection

Spotted cDNA microarraysSpotted cDNA microarraysAdvantages• Lower price and flexibility• Simultaneous comparison of two related

biological samples (tumor versus normal, treated versus untreated cells)

• ESTs allow discovery of new genes

Disadvantages• Needs sequence verification• Measures the relative level of expression

between 2 samples

Data Pre-processingData Pre-processing

Filtering – Background subtraction – Low intensity spots– Saturated spots – Low quality spots (ghost spots, dust

spots etc)

Normalization– Housekeeping genes/ control genes

Affymetrix Software for Microarray Data Analysis

• Microarray Suite 5 • Micro DB • Data Mining Tool (DMT)• NetAffx

Affymetrix Microarray Suite - Data AnalysisAffymetrix Microarray Suite - Data Analysis

Absolute Analysis –whether transcripts are Present or not (uses data from one probe array experiment).

Comparison Analysis –determine the relative change in transcripts (uses data from two probe array experiments).

Intensities for each experiment are compared to a baseline/control.

Microarray data analysisMicroarray data analysis

Scatter plots

• Intensities of experimental samples versus normal samples

• Quick look at the changes and overall quality of microarray

Normal vs. NormalNormal vs. Normal Normal vs. TumorNormal vs. Tumor

Lung Tumor: Lung Tumor: Up-RegulatedUp-Regulated

Lung Tumor: Lung Tumor: Down-RegulatedDown-Regulated

Microarray data analysisMicroarray data analysis

Supervised versus unsupervised analysis

– Clustering: organization of genes that are similar to each other

– Statistical analysis: how significant are the results?

Hierarchical clusteringHierarchical clustering • Unsupervised: no assumption on

samples

• The algorithm successively joins gene expression profiles to form a dendrogram based on their pair-wise similarities.

Cluster analysis of genes in G1 and G2

Chaudhry et. al., 2002

Publicly Available Softwares

GenMAPP

Visualize gene expression data on maps representing biological pathways and groupings of genes.

Microarray ApplicationsMicroarray Applications• Identify new genes implicated in disease progression and

treatment response (90% of our genes have yet to be ascribed a function)

• Assess side-effects or drug reaction profiles

• Extract prognostic information, e.g. classify tumors based on hundreds of parameters rather than 2 or 3.

• Identify new drug targets and accelerate drug discovery and testing

• ???

Microarray Technology - ApplicationsMicroarray Technology - Applications

• Gene Discovery-– Assigning function to sequence– Discovery of disease genes and drug targets– Target validation

• Genotyping– Patient stratification (pharmacogenomics)– Adverse drug effects (ADE)

• Microbial ID

The List Continues To Grow….

Microarray FutureMicroarray Future

• Must go beyond describing differentially expressed genes

• Inexpensive, high-throughput, genome- wide scan is the end game for research applications

• Protein microarrays (Proteomics)

Microarray FutureMicroarray Future

• Publications are now being focused on biology rather than technology

• SNP analysis –Faster, cheaper, as accurate as sequencing–Disease association studies–Population surveys

• Chemicogenomics–Dissection of pathways by compound application–Fundamental change to lead validation

Microarray FutureMicroarray Future

• Diagnostics– Tumor classification– Patient stratification– Intervention therapeutics

ConclusionConclusion

• Technology is evolving rapidly.• Blending of biology, automation, and

informatics.• New applications are being pursued

– Beyond gene discovery into screening, validation, clinical genotyping, etc.

• Microarrays are becoming more broadly available and accepted.– Protein Arrays– Diagnostic Applications

W.W.W resourcesW.W.W resources

• Complete guide to “microarraying” http://cmgm.stanford.edu/pbrown/mguide/

• http://www.microarrays.org– Parts and assembly instructions for printer and

scanner;– Protocols for sample prep;– Software;– Forum, etc.

• Animation: http://www.bio.davidson.edu/courses/genomics/chip/chip.html