Transcript Mosaic Analysis

Genomics I:
The Transcriptome
RNA Expression Analysis
Determining genomewide RNA
expression levels
Contents
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Genomewide RNA expression analysis
Northern blotting
Macroarrays
Microarrays
Making microarrays
Hybridization to microarrays
Microarray experiments
SAGE
Real-time PCR
Genomewide expression
analysis
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Goal: to measure RNA levels of all genes
in genome
RNA levels vary with the following:
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Cell type
Developmental stage
External stimuli
Time and location of expression provide
useful information as to gene function
Genomics expression analysis
methods
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Microarrays
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SAGE (Serial Analysis of Gene
Expression)
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Sequence fragments of cDNAs
MPSS (Massively Parallel Signature
Sequencing)
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Hybridization based
Combines hybridization and sequencing
Real-time PCR
Hybridization
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Measurements of RNA abundance by
microarrays based on hybridization
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Between complementary strands of RNA and
DNA
Or two complementary DNA strands
Similar in principle to RNA blot (Northern
blot)
Northern blot
–
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Electrophoresis of RNA
through gel
Transfer of RNA to solid
support
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Nylon or nitrocellulose
Intensity of
hybridization signal
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Approximately equal to
amount of RNA
gel
+
Hybridization issues
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RNA integrity must be verified
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Probe must be in excess of bound RNA
Hybridization kinetics govern reaction
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If RNA degraded, hybridization not quantitative
Hybridization must be for a sufficient time to
allow probe to find target RNA
Comparison between samples requires
loading control
Macroarray Analysis
Macroarray Analysis
Microarray Analysis of Transcription
Animation
Northern blots vs. microarrays
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Global expression
analysis: Northern
blot
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Limited by number of
probes that can be
used simultaneously
Global expression
analysis: microarrays
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RNA levels of every
gene in the genome
analyzed in parallel
target –
loading –
control
Basics of microarrays
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DNA attached to solid
support
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RNA is labeled
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Glass, plastic, or nylon
Usually indirectly
Bound DNA is the
probe
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Labeled RNA is the
“target”
Microarray hybridization
samples
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Usually comparative
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Ratio between two
samples
Examples
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Tumor vs. normal
tissue
Drug treatment vs. no
treatment
Embryo vs. adult
mRNA
cDNA
DNA
microarray
Two major types of microarrays
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cDNA arrays- PCR product corresponding
to a portion of a cDNA is immobilized on
the slide
oligonucleotide arrays- oligonucleotide
complementary to transcript is synthesized
on slide or immobilized on the slide
How microarrays are made:
spotted microarrays
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DNA mechanically placed on glass slide
Need to deliver nanoliter to picoliter volumes
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Too small for normal pipetting devices
Robot “prints,” or “spots,” DNA in specific places
DNA spotting I
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DNA spotting usually
uses multiple pins
DNA in microtiter
plate
DNA usually PCR
amplified
Oligonucleotides can
also be spotted
DNA spotting II
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Pins dip into DNA solution in microtiter wells
Robot moves pins with DNA to slides
Robot “prints” DNA onto slide
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Same spots usually printed at different locations
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DNA sticks to slide by hydrostatic interactions
Serves as internal control
Pins washed between printing rounds
Hundreds of slides can be printed in a day
Commercial DNA spotter
How microarrays are made:
Affymetrix GeneChips
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Oligonucleotides synthesized on silicon chip
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One base at a time
Uses process of photolithography
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Developed for printing computer circuits
Affymetrix GeneChips
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Oligonucleotides
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Oligonucleotides for each gene selected by
computer program to be the following:
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Usually 20–25 bases in length
10–20 different oligonucleotides for each gene
Unique in genome
Nonoverlapping
Composition based on design rules
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Empirically derived
Photolithography
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Light-activated chemical
reaction
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Custom masks
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For addition of bases to
growing oligonucleotide
Prevent light from reaching
spots where bases not
wanted
Mirrors also used
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NimbleGen™ uses this
approach
lamp
mask
chip
Example: building oligonucleotides
by photolithography
light
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Want to add nucleotide G
Mask all other spots on
chip
Light shines only where
addition of G is desired
G added and reacts
Now G is on subset of
oligonucleotides
Example: adding a second base
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Want to add T
New mask covers spots
where T not wanted
Light shines on mask
T added
Continue for all four bases
Need 80 masks for total
20-mer oligonucleotide
light
Ink-jet printer microarrays
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Ink-jet printhead draws up DNA
Printhead moves to specific location on solid
support
DNA ejected through small hole
Used to spot DNA or synthesize oligonucleotides
directly on glass slide
Use pioneered by Agilent Technologies, Inc.
Comparisons of microarrays
Comparison of microarray
hybridization
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Spotted microarrays
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Competitive hybridization
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Two labeled cDNAs hybridized to same slide
Affymetrix GeneChips
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One labeled RNA population per chip
Comparison made between hybridization
intensities of same oligonucleotides on
different chips
Target labeling: fluorescent
cDNA
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cDNA made using
reverse transcriptase
Fluorescently labeled
nucleotides added
Labeled nucleotides
incorporated into
cDNA
Target labeling: cRNA + biotin
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cDNA made with
reverse transcriptase
Linker added with
T7 RNA polymerase
recognition site
T7 polymerase
added and biotin
labeled RNA bases
Biotin label
incorporated into
cRNA
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Labels
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Cy3 and Cy5
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Fluoresce at different wavelengths
Used for competitive hybridization
Biotin
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Binds to fluorescently labeled avidin
Used with Affymetrix GeneChips
Spotted-microarray hybridization
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Control and experimental cDNA labeled
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One sample labeled with Cy3
Other sample labeled with Cy5
Both samples hybridized together to
microarray
Relative intensity determined using
confocal laser scanner
Scanning of microarrays
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Confocal laser
scanning microscopy
Laser beam excites
each spot of DNA
Amount of
fluorescence detected
Different lasers used
for different
wavelengths
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Cy3
Cy5
laser
detection
Analysis of hybridization
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Results given as ratios
Images use colors:
Cy3 = Green
Cy5 = red
Yellow
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Yellow is equal
intensity or no change
in expression
Example of spotted microarray
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RNA from irradiated
cells (red)
Compare with
untreated cells (green)
Most genes have little
change (yellow)
Gene CDKN1A: red =
increase in expression
Gene Myc: green =
decrease in expression
CDKNIA
MYC
Analysis of cell-cycle regulation
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Yeast cells stopped at
different stages of cell
cycle
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G1, S, G2, and M
RNA extracted from
each stage
Control RNA from
unsynchronized
culture
Results of yeast cell-cycle analysis
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800 genes identified whose expression
changes during cell cycle
Grouped by peak expression
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Four different treatments used to
synchronize cells
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M/G1, G1, S, G2, and M
All gave similar results
Results from Spellman et al., 1998; Cho
et al., 1998
Cell-cycle regulated genes
Alpha
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Each gene is a line on
the longitudinal axis
Treatments in
different panels
Cell-cycle stages are
color coded at top
Vertical axis groups
genes by stage in
which expression
peaks
cdc15
cdc28
Elu
M/G1
G1
S
G2
M
Brown and Botstein, 1999
Affymetrix GeneChip experiment
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RNA from different types of brain tumors
extracted
Extracted RNA hybridized to GeneChips
containing approximately 6,800 human
genes
Identified gene expression profiles specific
to each type of tumor
Profiling tumors
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Image portrays gene
expression profiles
showing differences
between different
tumors
Tumors:
MD (medulloblastoma)
Mglio (malignant glioma)
Rhab (rhabdoid)
PNET (primitive
neuroectodermal tumor)
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Ncer: normal cerebella
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Gene expression
differences for
medulloblastoma
correlated with response
to chemotherapy
Those who failed to
respond had a different
profile from survivors
Can use this approach to
determine treatment
60 different samples
Cancer diagnosis by microarray
Analysis of microarray results
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Inherent variability: need for repetition
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Analysis algorithms
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Biological and technical replicates
Based on statistical models
Means of generating hypotheses that need
to be tested
SAGE I
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Serial analysis of gene expression
Concept: sequence a small piece of each
cDNA in a library
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Gives measure of abundance of each RNA
species
Method
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Cut off “tag” from each cDNA
Ligate tags together into a concatemer
Sequence the concatemer
SAGE II
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Cleave cDNAs with fourbase cutter restriction
enzyme
AAAAAAA
TTTTTTT
GTAC
AAAAAAA
TTTTTTT
Ligate adapters
containing site for typeIIs restriction enzyme
CATG
GTAC
AAAAAAA
TTTTTTT
Cut 14 base pairs from
recognition site
CATG
GTAC
AAAAAAA
TTTTTTT
SAGE III
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Ligate on adapters with
restriction sites
Cut with two restriction
enzymes to release 26
base pair tag
Ligate tags together
into ~500 base pair
concatemer
CATG
GTAC
GGTCAC
CCAGTG
CATG
GTAC
GGTCAC
CCAGTG
CATG
GTAC
GGTCAC
CCAGTG
CATG
GTAC
SAGE IV
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Sequence the concatemers
Identify tag borders
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Size of tag and restriction-enzyme sites
Compare tag sequences to database
Abundance of tag is measure of abundance
of that RNA species
Real-time PCR
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Sensitive means of
measuring RNA
abundance
Not genomewide: used to
verify microarray results
TaqMan method uses
fluorescently tagged
primers
Fluorescent tag released
by Taq polymerase
Real-time PCR readout
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The readout of a realtime PCR reaction is a
set of curves
The curves indicate
the PCR cycle at
which fluorescence is
detected
Each cycle is twice
the amount of the
previous cycle
Genomic analysis of gene
expression
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Methods capable of giving a “snapshot” of RNA
expression of all genes
Can be used as diagnostic profile
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Example: cancer diagnosis
Can show how RNA levels change during
development, after exposure to stimulus, during
cell cycle, etc.
Provides large amounts of data
Can help us start to understand how whole
systems function