Some aspects of the design and analysis of gene expression microarray experiments

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Introduction to Microarray Analysis and
Technology Dave Lin - November 5, 2001
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Overview

• Why Biologists care about Genomics

• Why statisticians/computer scientists
• may care about genomics
• Preprocessing issues
• Sources of variability in constructing
• microarrays
• Postprocessing issues
• Analysis of data

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What makes one cell different from another?
liver vs. brain Cancerous vs. non-cancerous Treat
ment vs. control
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Old Days 100,000 genes in mammalian
genome each cell expresses 15,000 of these
genes each gene is expressed at a different
level estimated total of 100,000 copies of
mRNA/cell 1-5 copies/cell - rare -30 of all
genes 10-200 copies/cell - moderate 200
copies/cell and up - abundant
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Cells can be defined by Complement of Genes
(which genes are expressed) How much of each
gene is expressed (quantity) What makes one cell
different from another? Try and find genes that
are differentially expressed Study the function
of these genes Find which genes interact with
your favorite gene
Extremely time-consuming. Huge amounts of effort
expended to find individual genes that may differ
between two conditions
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Genomics. Almost useless term-defines many
different concepts and applications. Microarray
s -massively parallel analysis of gene
expression -screen an entire genome at
once -find not only individual genes that
differ, but groups of genes that
differ. -find relative expression level
differences -how quantitative can they be?
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Microarrays- Based on old technique many
flavors- majority are of two essential
varieties cDNA Arrays printing on glass
slides miniaturization, throughput fluoresce
nce based detection Affymetrix Arrays in
situ synthesis of oligonucleotides will not
consider Affymetrix arrays further.
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Building the Chip
PCR PURIFICATION and PREPARATION
MASSIVE PCR
Full yeast genome 6,500 reactions
IPA precipitation EtOH washes 384-well format
PRINTING
The arrayer high precision spotting device
capable of printing 10,000 products in 14 hrs,
with a plate change every 25 mins
PREPARING SLIDES
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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Fabrication of Spotted Arrays
20,000 Precipitations
20,000 PCR reactions
Arrayed Library Normalized/Subtracted
Consolidate for printing
20,000 resuspensions
Spot on Glass Slides
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Micro Spotting pin
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Hybing the Chip
ARRAY HYBRIDIZATION
Cy3 and Cy5 RNA samples are simultaneously
hybridized to chip. Hybs are performed for 5-12
hours and then chips are washed.
DATA ANALYSIS
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.
PROBE LABELING
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.
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Labeling of RNAs with Cy3 or Cy5 Two general
methods -Dye conjugated nucleotide -Amino-ally
l indirect labeling
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Direct labeling of RNA
RNA
AAAAAAA
cDNA
TTTTTTTT
CCAACCTATGG
T
Cy5-dUTP

or
cDNA synthesis
T
Cy3-dUTP
GGTTGGATACC
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Indirect labeling of RNA
AAAAAAA
TTTTTTTT
CCAACCTATGG
T
Modified nucleotide
cDNA synthesis
GGTTGGATACC
Cy3
GGTTGGATACC
addition
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Dye effect issues
Direct method Unequal incorporation of Cy5 vs.
Cy3 Very poor overall incorporation of
direct-conjugated nucleotide more starting RNA
for labeling. Indirect method Presumably less
bias in initial incorporation of activated
nucleotide, but not clear if more or less dye is
added Both Methods Cy3 fluoresces more
brightly than Cy5 labeling is very highly
sequence dependent
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Micrograph of a portion of hybridization probe
from a yeast mciroarray (after hybridization).
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Layout of the cDNA Microarrays

• Sequence verified, normalized mouse cDNAs
• 19,200 spots in two print groups of 9,600 each
• 4 x 4 grid, each with 25 x24 spots
• Controls on the first 2 rows of each grid.

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Practical Problems 1

• Comet Tails
• Likely caused by insufficiently rapid immersion
  of the slides in the succinic anhydride blocking
  solution.

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Practical Problems 2
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Practical Problems 3

• High Background
• 2 likely causes
• Insufficient blocking.
• Precipitation of the labeled probe.
• Weak Signals

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Practical Problems 4
Spot overlap Likely cause too much
rehydration during post - processing.
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Practical Problems 5
Dust
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Pin-specific printing differences
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Normalization - lowess

• Global lowess
• Assumption changes roughly symmetric at all
  intensities.

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Normalisation - print-tip-group
Assumption For every print group, changes
roughly symmetric at all intensities.
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Pre-processing Issues
-Definition of what a real signal is what is a
spot, and how to determine what should be
included in the analysis? -How to determine
background local (surrounding spot) vs. global
(across slide) -How to correct for dye
effect -How to correct for spatial effect e.g.
print-tip, others -How to correct for differences
between slides e.g. scale normalization
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Experimental Design Issues What is the best
means of performing the experiment To obtain the
desired answer? Biologists assumptions and
statisticians differ. Biologist
viewpoint make everything exactly the same so
that differences will stand out Statistician
viewpoint make everything as random as
possible so that real trends will stand out
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Most biologists will ask- what are the
differences between two samples? -implicit
questions associated with microarrays- What is
the best way to determine this? e.g. Design
replicates conditions. How do I obtain the most
reliable results? e.g. measurements,
normalization How do I determine what a
significant difference is? Do I care about
subtle changes, or just the extremes? How is
information best extracted? Is correlation
useful? What type of clustering? How is
information combined? How do you model the
interactions of 1000s of genes
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Design Two Ways to Do the Comparisons
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Advantages of Our Design

• Lower variability
• Increased precision
• Increase in measurement of expression -gt
  increased precision

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