DNA Microarray

Mehran Haidari, PhDHospital for Sick ChildrenUniversity of Toronto

Approaches for Characterizing Differential Gene Expression

Low-throughput or Single Gene Methods

High-throughput or Large-Scale Methods

The Hybridization of Complementary Strands of DNA/RNA Is the Underlying Principle of All Methods of Differential Gene Expression.

Single Gene Methods

Northern Blotting, cumbersome,time-consuming

Nuclease protection, at least 10 fold more sensitive

Quantitative RT-PCR, state of art

High-throughput Methods

Serial Analysis of Gene Expression (SAGE)

Rapid Analysis of Gene Expression (RAGE)

Representational Difference Analysis (RDA)

Suppression Subtractive Hybridization (SSH)

Differential screening (plus/minus screening)

Differential Display (DD)

DNA Microarray

What is DNA Microaray?

Microarray pioneers:Pat Brown and Schena

A large number of genes deposited onto a glass slide (large scale dot blot)

The RNA sample is RT with simultaneous incorporation of label, resulting in labeled cDNA.

Microarray slides serve as hybridization targets for labeled cDNA

Analysis of Gene Expression

Monitoring Changes in Genomic DNA

Gene Discovery, Sequencing and Pathway Analysis

When to use Microarray

Analysis of Gene Expression

1- Different tissues or at different developmental states

2- Normal or diseased states3- Exposure to drugs or different physiological conditions

Monitoring Changes in Genomic DNA

Hybridization to oligonucleotid is sensitive to detect single-nucleotide mismatches

Single Nucleotide Polymorphisms (SNPs)

High Density Oligonucleotide Array

Cancer cells typically exhibit genomic instability

Basic Steps in Performing a DNA Microarray Experiments

1- Processing cDNA clones to generate print-ready material

2-Printing cDNA clones (or oligonucleotide) onto a substrate

3-Sample RNA isolation

4-Preparation of the probe (e.g. cDNA synthesis and labeling, RT reaction)

5- Hybridization of labeled probe DNA to the DNA arrayed on the substrate

6-Image acquisition, image analysis and data analysis

What to spot

As many known genes as possible

Genes that are most relevant

A combination of both approaches

Publicity available clones(IMAGE)

In_house derived (SSH)

Custom made/purchased libraries

Detailed Protocols

Stanford University

Albert Einstein College of Medicine

NHGRI

Cold Spring Harbor Laboratory

Collection of Protocols

TIGR Protocols

www.cmgm.stanford.edu/pbrown/

www.sequence.aecom.yu.edu/bioinf/microarray/protocol.html

www.nhgri.gov/DIR/LCG/15K/HTML/protocol.html

www.nucleus.cshl.org/wigler

www.protocol-online.net/molbio/DNA/dna_microarray.html

www.tigr.org/tdb/microarray

Microarray Fabrication Technologies

In Situ Synthesis of Nucleic Acid (Chip ,GeneChip,oligonucleotide array)

15-20 different 25-mer oligonucleotides

Exogenous Deposition of cDNA (cDNA, spotted array)

Single DNA fragments, greater 0.5 Kb

Common Approaches for Microarray Fabrication

1-Contact printing (Patrick O Brown,Stanford University)

2- Non-Contact Printing (Pin and Ring, Bubble Jet, Ink Jet)

3- Photolithography (Affymetrix, Oligonucleotide Microarray)

http://www.affymetrix.com/technology/tech_probe.html

Contact Printing

Non-Contact Printing

Photolithography

Affymetrix Technology

Two basic substrates commonly used for cDNA printingare glass and membrane filters

Chemically treated microscope glass slides are the most widely used support

Microarray, Microscope Slide,80000 Spots, 10000-20000 Spots

Macroarray, Nylon Membrane, 500,-18000 Spots

Micro or Macro

RNA Preparation

No difference between total RNA or mRNA

Type of tissue might have profound effect on extraction process. 100 -200 µg of RNA is need/slide

Laser capture microdissection (LCM) , incorporation of a PCR step

Sample Labeling

Most microarray utilize two fluorophores,Cyanine3(Green emission) and Cyanine5 (Red emission)

They have different size and different abilityfor incorporation in cDNA

A single round of transcription is used to generate a labeled cDNA probe (RT-PCR)

Data AnalysisSeldom more than two replicates of each experiment

Normalization

First step is during scanning, when sensivity of detection is adjusted by the laser voltage

Gene expression value can be expressed relative to the expression of housekeeping genes

In the absence of control genes, normalization to the medianmicroarray value is popular

Analyzed gene changes are often expressed as a fold increaseeither greater than twofold or less than 0.5 fold (DeRisi)

How Much is Significant???

With a large number of microarrays, small changes can be statistically valid

Elcock et al. detected 1.1 fold changes with %95 confidence interval wheneach experimental sample was hybridized to seven microarray slides (with two replicate spots for each gene)

Derisi et al.Nat Genet 1996:14:457-60

Housekeeping genes

These are genes that are expressed constitutively and their level of expression is thought to be stable, regardless of the sample used ( Actin, Cyclophilin, GAPDH)

DeRisi used 90 housekeeping genes and found that changes thatwere <0.5 and > 2.4 were acceptable

Actin is one of the most commonly used housekeeping genes and it has been shown to be downregulated in heat shock experiments

In fact, there is an appreciable amount of literature available to suggest that there is no such thing as housekeeping gene

DNA microarray represents a developing technology, there remain substantial obstacles in the design and analysis of these microarray

There are no globally accepted rules or standardsfor performing controlled microarray experiments

A good experiments include more control component then the real comparison

Accuracy and Precision

Quality Control of DNA Microarray

Down-Scaling of an experiment makes it generally sensitive to external and internal fluctuation

Replication of each experiments on multiple array

Dual labeling, swapping the dyes for control and treated sample

Using a large number of controls on every array

ControlsmRNA from genes that are not homologous to the organism understudy (Arabidopsis)

cDNA from the organism with high, medium and low expression represented on the array (sensivity)

Cold DNA (e.g., calf thymus DNA, yeast tRNA)is added to block nonspecific annealing

Spots of DNA from another organism whose mRNA is not represented in the sample (Background)

Total genomic DNA or cDNA clones of common contaminant such as E.Coli and yeast are represented in the array to monitor for contamination

C.C Liew,(1994) sequenced 3500 ESTs representing 3100 cDNA from adult human heart(First cardiovascular catalogue of genes)

The number of cardiovascular ESTs increased to 85,000 (1997)

The latest number(2001) is 111,224 cardiovascular ESTs

The largest cardiovascular cDNA microarray constructed (10,368 ESTs)

Cardiovascular ESTsChoong-Chin Liew, Director, The Cardiovascular Genome Unit,Harvard Medical School

The number of genes encoded by the Human Genome has been estimated to be 32,000 - 38,000.

Between 21,000 - 27,000 genes are expressed in the cardiovascular system

Lack of information

No cDNA Library for Atherosclerotic plaques

Only 5% of total ESTs deposited in GeneBank are derived from cardiovscular tissue

ESTs from cardiovascular tissues or cell type or from diseased specimens remain limited

                                            

  

Cardiovascular EST data from most model organisms are almost nonexistent

The construction of cardiovascular gene databases at different stages of pathology will cast light on the complex genetic mechanisms underlying disease of cardiovascular system

DNA microarray technology is still in its infancy

DNA microarray in atherosclerosis is not born yet (or at most is premature)

Premature

B.C.G. FABER did the first study dealing with differential gene expression in whole-mount specimens of rupture plaques using macroarray

Suppression Subtractive Hybridization (SSH) technique isolates low abundant sequence that might not be isolated by use of microarray technology

Mammalian mRNA population

20% Abundant transcript (1000-12000 copies/cell)

25% Medium abundant (100-1000 copies/cell)

% 50 small number copies (< 13 copies/cell)

Mammalian mRNA encoding proteins that regular cellular behavior are expressed at low abundance

Circ Rec 2001.89;547-554 University of Mastricht, Netherland

SSH3 ruptured plaques3 stable plaques

Forward reaction n=3000

Reverse reaction n=2000

Macro array n=500

Sequencing n=25

RT-PCR analysis n=3

RNA in situ hybridization n=1

Immunohistochemistry n=1

> two fold difference

Perilipin was the known gene that upregulated (confirmed by RT-PCR) 8 of 10 ruptured plaques expressed prelipin while expression was absent in 10 stable plaque

Prelipin is a protein which present on the surface layer of intracellular lipid droplets in adipocyte and prevent lipolysis

actin was down regulated in ruptured plaques

Prelipin is unlikely to be the sole marker of rupture

The author used only 10% of differentially expressed gene for doing macroarrayA large effort at macroarray and then sequencing would have yield more differences

An alternative would be to hybridized the subtractand against a large array

Other alternative is the isolation of cell type-specific genes (LCM) rather than plaque-type-specific genes

K.j.Haley et al. treated cultured Human aortic SMC with TNF and used DNA microarray with 8600 genes to monitor gene expression

Marked increase in eotaxin confirmed with northern blotting

Immunohistochemical analysis demonstrated overexpression of eotaxin and its receptor in the human atheroma (SMC)

Circulation;2000:102:2185-2189 Boston-Harvard

McCaffrey et al. compared transcript profile of fibrous cap vs. adjacent media of 13 patients, using macroarray (membrane 588 known genes)

Early growth response gene(Egr-1) was highly expressed in lesion (confirmed by RT-PCR)

Many Erg-1 inducible genes including PDGF , TGF- and ICAM-1 were also strongly elevated in the lesion

ACTIN and GAPDH were use as housekeeping gene

J.C.I 2000,105:653-662 Cornell University

L.D Adams, S.M Schwartz, University of Washington

Adams et al. Compared gene expression of media of aorta and vena cava, using cDNA microarray of 4048 known genes

68 genes had consistent elevation in message expression of the aorta

The most differentially gene was Regulator of G protein Signaling (RGS5)

Northern analysis and in situ hybridization were used to confirm the results

Circulation Research 2000.8.623

R.M Lawn et al. examined the response of macrophages to exposure to oxidized LDL, using microarray containing 10000 Human genes

268 genes were found to be at least twofold regulated

Real Time RT-PCR was used to confirm the results

Orphan nuclear receptors (PPAR, LXR and RXR) and ABC1 wereamong genes which unregulated after exposure

J.B.C 2000:275;48, 37324-37332

L.A Mcintire et al. identified 52 genes with altered expression under shear stressUsing DNA microarray in primary human umbilical vein endothelial cells

Significant increases in mRNA levels for 32 and significant decreases in expression for 20 genes were reported

The most enhanced genes were cytocromes P45 1A1 and 1B1 and human prostaglandin transporter

Most dramatically decreased were connective tissue growth factor and endotheline-1

PNAS2001, 98:8955-8960 Rice University

Rajeevan et al. estimated that % 30 of microarray results are false-positive

Rajeevan et al. J Mol Diag 2001-3-26-31

Microarray findings should be confirmed,at least by one of the low-throughput gene expression methods

Northern Blotting

Nuclease protection

Quantitative RT-PCR

Many genes are expressed constitutively and regulation of their function is at the transnational or posttranslational(ApoB ,CFTR, TCR)

To date, there has been a relatively poor correlation between gene and protein expression.

It is likely that global proteome analysis provides a betterrepresentation of the phenotype than does gene expression analysis