Transcript No Slide Title - University of Vermont

DNA Microarrays
M. Ahmad Chaudhry, Ph. D.
Director Microarray Facility
University of Vermont
Outline of the lecture
• Overview of Micoarray Technology
• Types of Microarrays
• Manufacturing
• Instrumentation and Softwares
• Data analysis
• Applications
Microarray Development
• Relatively young technology
• Widely adopted
• Mainly used in gene discovery
Evolution & Industrialization
• 1994- First cDNAs arrays
were developed at
Stanford University.
• 1996- Commercialization
of arrays
• 1997-Genome-wide
Expression Monitoring in
S. cerevisiae
What are Microarrays?
• 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”
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?
• 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
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)
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 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 Probes
Set of oligos to be synthesized is defined, based on its ability to
hybridize to the target genes of interest
5’
3’
Sequence
Probes
Perfect Match
Mismatch
Chip
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 Synthesis
Manufacturing Process
Probe arrays are manufactured by light-directed chemical
synthesis process which enables the synthesis of hundreds of
thousands of discrete compounds in precise locations
Lamp
Mask
Chip
Affymetrix Wafer and Chip Format
20 - 50 µm
20 - 50 µm
Millions of identical
oligonucleotide
probes per feature
49 - 400
chips/wafer
1.28cm
up to ~ 400,000 features/chip
Creating Targets
mRNA
Reverse Transcriptase
cDNA
in vitro transcription
cRNA
Target
RNA-DNA Hybridization
Targets
RNA
probe sets
DNA
(25 base oligonucleotides of known sequence)
Non-Hybridized Targets are Washed Away
Targets
(fluorescently tagged)
“probe sets” (oligo’s)
Non-bound ones are washed away
Target Preparation
B
Biotin-labeled
transcripts
B
B
B
B
Fragment
(heat, Mg2+)
B
B
B
Fragmented cRNA
IVT
AAAA
mRNA
(Biotin-UTP
Biotin-CTP)
Wash & Stain
Scan
cDNA
Hybridize
(16 hours)
®
GeneChip Expression Analysis
Hybridization and Staining
Array
Hybridized Array
cRNA Target
Streptravidinphycoerythrin
conjugate
Instrumentation
Affymetrix GeneChip System
3000-7G Scanner
450 Fluidic Station
640 Hybridization Oven
Currently Available GeneChips
B. subtilis
Plasmodium/Anopheles Genome Array
Barley Genome Array
Porcine Genome Array
Bovine Genome Array
Rat Genome Arrays
C. elegans Genome Array
Rice Genome Array
Canine Genome Array
Soybean Genome Array
Chicken Genome Array
Sugar Cane Genome Array
Drosophila Genome Arrays
Vitis vinifera (Grape) Array
E. coli Genome Arrays
Wheat Genome Array
Human Genome Arrays
Xenopus laevis Genome Array
Maize Genome Array
Yeast Genome Arrays
Mouse Genome Arrays
Zebrafish Genome Array
P. aeruginosa 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 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
steel
Spotted arrays
spotting pin
chemically modified slides
384 well source
plate
1 nanolitre spots
90-120 um diameter
The process
Building the chip:
MASSIVE PCR
PCR PURIFICATION
and PREPARATION
PREPARING SLIDES
RNA
preparation:
CELL CULTURE
AND HARVEST
PRINTING
Hybing the
chip:
POST PROCESSING
ARRAY HYBRIDIZATION
RNA ISOLATION
DATA ANALYSIS
cDNA PRODUCTION
PROBE LABELING
Building the chip
Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)
Spot as microarray
on glass slides
PCR amplification
Directly from colonies with
SP6-T7 primers in 96-well
plates
Consolidate into
384-well plates
Sample preparation
Hybridization
Binding of cDNA target samples to cDNA probes on the slide
Hybridize for
5-12 hours
Hybridization chamber
3XSSC
HYB CHAMBER
ARRAY
LIFTERSLIP
SLIDE
LABEL
SLIDE LABEL
• Humidity
• Temperature
• Formamide
(Lowers the Tm)
Expression profiling with DNA microarrays
cDNA “B”
Cy3 labeled
cDNA “A”
Cy5 labeled
Laser 1
Hybridization
Laser 2
Scanning
+
Analysis
Image Capture
Image 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 analysis
GenePix
Image 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
Data
Analysis &
Modelling
Microarray
Life Cycle
Sample
Preparation
Microarray
Detection
Microarray
Reaction
Spotted cDNA microarrays
Advantages
• 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-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 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 analysis
Scatter plots
• Intensities of experimental samples versus
normal samples
• Quick look at the changes and overall quality of
microarray
Normal vs. Normal Normal vs. Tumor
Lung Tumor:
Up-Regulated
Lung Tumor:
Down-Regulated
Microarray data analysis
Supervised versus unsupervised analysis
– Clustering: organization of genes that are
similar to each other
– Statistical analysis: how significant are the
results?
Hierarchical 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 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 - 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 Future
• Must go beyond describing differentially
expressed genes
• Inexpensive, high-throughput, genomewide scan is the end game for research
applications
• Protein microarrays (Proteomics)
Microarray 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 Future
• Diagnostics
– Tumor classification
– Patient stratification
– Intervention therapeutics
Conclusion
• 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 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