Macroarray technology

1
Macroarray technology
2
Overview
Lecture One How to obtain cDNA clones to spot on
macroarrays How macroarrays are spotted
Lecture Two Normalization and analysis of
data Demonstration of macroarray spotting
3
Macroarray vs. microarray
Macro array Hundreds of cDNA clones spotted on
array
Micro array Thousands of cDNA clones spotted on
array
4
Getting clones to spot on macroarray
WHY???
5
Getting clones to spot on macroarray
WHY??? cDNA clones are not readily available
from non-traditional animal or plant model systems
6
Getting clones to spot on macroarray
Want to examine expression of genes in livers
of fish that are involved in detoxification or Ex
amine expression of genes involved in hypoxia in
sea corals. or _______
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Getting clones to spot on macroarray
Want to examine expression pattern of 3-4 genes
that you already have
8
Getting clones to spot on macroarray
Want to examine expression pattern of 3-4 genes
that you already have
Slot blots Northerns RT PCR (Taq man)
X
Macroarrays
9
Getting clones to spot on macroarray
Want to examine expression pattern of 3-4 genes
that you already have
Slot blots Northerns RT PCR (Taq man)
X
Macroarrays (15 or more clones)
10
Getting 15 or more clones to spot on macroarray
Directed approach vs. non-directed approach
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Getting 15 or more clones to spot on macroarray
Directed approach
Design degenerate primers to desired genes (ex.
Carbonic Anhydrase) using known sequences in the
database.
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Directed approach
Getting clones to spot on macroarray
Design degenerate primers to desired genes (ex.
Carbonic Anhydrase) using known sequences in the
database.
? Your species ?
Mouse
Human
Drosophila
Conserved region
Conserved region
Unique region
13
Directed approach
Getting clones to spot on macroarray
Design degenerate primers to desired genes (ex.
Carbonic Anhydrase) using known sequences in the
database.
? Your species ?
Mouse
Human
Drosophila
Conserved region
Conserved region
Unique region
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Directed approach
Getting clones to spot on macroarray
Advantages
-Can target selective genes
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Directed approach
Getting clones to spot on macroarray
Advantages
-Can target selective genes
Disadvantages
-Designing individual primer sets and testing
them is a time consuming process.
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Non-Directed approach
Getting clones to spot on macroarray
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Non-Directed approach
Getting clones to spot on macroarray
Want to examine expression of any genes in
livers of fish that are involved in
detoxification.
18
Non-Directed approach
Getting clones to spot on macroarray
Want to examine expression of any genes in
livers of fish that are involved in
detoxification. TECHNIQUES cDNA
libraries Differential display analysis Subtractiv
e hybridization
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cDNA libraries
Tissue
Modified from D. Moraga slide
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cDNA libraries
Tissue
Isolation
mRNA
Modified from D. Moraga slide
21
cDNA libraries
Tissue
Isolation
mRNAs
Reverse Transcription
cDNAs
Modified from D. Moraga slide
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cDNA libraries
Population of different cDNAs
Left
Right
Arms of phage cloning vector
Modified from D. Moraga slide
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cDNA libraries
Population of different cDNAs
Left
Right
Arms of phage cloning vector
Modified from D. Moraga slide
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cDNA libraries
Population of different cDNAs
Left
Right
Arms of phage cloning vector
In vitro packaging
Modified from D. Moraga slide
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cDNA libraries
E. Coli
Modified from D. Moraga slide
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cDNA libraries
E. Coli
Plate
Modified from D. Moraga slide
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cDNA libraries
E. Coli
Plate
Modified from D. Moraga slide
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cDNA libraries
E. Coli
Plate
-In vivo excision -Sequence -Spot on array
Modified from D. Moraga slide
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cDNA libraries
Advantages
-If you already have a cDNA library, you can
easily obtain hundreds of clones to spot onto a
macroarray.
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cDNA libraries
Advantages
-If you already have a cDNA library, you can
easily obtain hundreds of clones to spot onto a
macroarray.
Disadvantages
-Generating a cDNA library is both time
consuming and expensive. -You may spot redundant
cDNAs onto the macroarray. -No guarantee
that any cDNA clones that you spot on the
macroarrays will be differential regulated.
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Differential Display Analysis
Genhunter
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Differential Display Analysis
Genhunter
mRNA
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
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Differential Display Analysis
Genhunter
mRNA
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Reverse transcribe
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Differential Display Analysis
Genhunter
mRNA
Anchor Primer (3)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Reverse transcribe
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Differential Display Analysis
Genhunter
mRNA
Anchor Primer (3)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Reverse transcribe
36
Differential Display Analysis
Genhunter
mRNA
Anchor Primer (3)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Reverse transcribe
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
37
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
PCR Amplify
labeled dNTPs
38
Anchor Primer (3)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
PCR Amplify
labeled dNTPs
39
Anchor Primer (3)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Arbitrary Primers (80)
PCR Amplify
labeled dNTPs
40
Anchor Primer (3)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Arbitrary Primers (80)
PCR Amplify
labeled dNTPs
X
Y
Z
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Anchor Primer (3)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Arbitrary Primers (80)
PCR Amplify
labeled dNTPs
X
Y
Z
Run on denaturing Polyacrylamide gel
42
Anchor Primer (3)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Arbitrary Primers (80)
PCR Amplify
labeled dNTPs
X
Y
Z
Run on denaturing Polyacrylamide gel
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(No Transcript)
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Constant
Induced
Induced
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Constant
Induced
Induced
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Gel extract
PCR amplify
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Gel extract
PCR amplify
Ligate in vector, transformminiprep and
sequence
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Gel extract
PCR amplify
Ligate in vector, transformminiprep and
sequence
Spot sequences on arrays
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Differential Display Analysis
Genhunter
Advantages
-Technology is fairly simple. -You increase your
probability that the clones that you
spot on the macroarrays will be differentially
regulated.
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Differential Display Analysis
Genhunter
Advantages
-Technology is fairly simple. -You increase your
probability that the clones that you
spot on the macroarrays will be differentially
regulated.
Disadvantages
-It would be fairly expensive to get hundreds of
differentially expressed clones. -Hard to
identify the sequences that you clone in
the data base using genehunter technology.
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Differential Display Analysis
Genhunter
Anchor Primer (3)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Arbitrary Primers (80)
52
Differential Display Analysis
Genhunter
Anchor Primer (3)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Arbitrary Primers (80)
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Differential Display Analysis
Qbiogene
Generate cDNA (similar to the Genehunter method)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
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Differential Display Analysis
Qbiogene
Generate cDNA (similar to the Genehunter method)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Restriction digest
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Differential Display Analysis
Qbiogene
Generate cDNA (similar to the Genehunter method)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Restriction digest
Ligate adapters
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Differential Display Analysis
Qbiogene
Generate cDNA (similar to the Genehunter method)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Restriction digest
Ligate adapters
57
Differential Display Analysis
Qbiogene
Generate cDNA (similar to the Genehunter method)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Restriction digest
Ligate adapters
58
Differential Display Analysis
Qbiogene
Generate cDNA (similar to the Genehunter method)
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Restriction digest
Ligate adapters
PCR Amplify
XYZ
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Differential Display Analysis
Qbiogene
Advantages
-Get sequences in the coding region.
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Differential Display Analysis
Qbiogene
Advantages
-Get sequences in the coding region.
Disadvantages
-Technology is NOT robust.
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Subtractive Hybridization Clontech
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Subtractive Hybridization
RNA from experimental tissue.
RNA from Control tissue.
Reverse transcription
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Subtractive Hybridization
RNA from experimental tissue.
RNA from Control tissue.
Reverse transcription
cDNA
cDNA
Digestion Addition of adapters Denature and mix
samples
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Subtractive Hybridization
RNA from experimental tissue.
RNA from Control tissue.
Reverse transcription
cDNA
cDNA
Digestion Addition of adapters Denature and mix
samples
Amt
cDNA pool
cDNA pool
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Subtractive Hybridization
RNA from experimental tissue.
RNA from Control tissue.
Reverse transcription
cDNA
cDNA
Digestion Addition of adapters Denature and mix
samples
Amt
cDNA subtracted out
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Subtractive Hybridization
RNA from experimental tissue.
RNA from Control tissue.
Reverse transcription
cDNA
cDNA
Digestion Addition of adapters Denature and mix
samples
PCR amplified, cloned, sequenced
Amt
cDNA subtracted out
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Subtractive Hybridization
Advantages
-Can generate hundreds of clones quickly.
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Subtractive Hybridization
Advantages
-Can generate hundreds of clones quickly.
Disadvantages
-Get redundant sequences. -Technology is slightly
expensive.
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Summary
Directed Degenerate primers Non-directed cDNA
library Differential display Subtractive
hybridization
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Strategy
Start with Differential Display or subtractive
hybridization
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Strategy
Start with Differential Display or subtractive
hybridization Fill in select genes with
degenerate primers
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Strategy
Start with Differential Display or subtractive
hybridization Fill in select genes with
degenerate primers and add Non-select genes
with cDNA library
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Overview
Lecture One Obtaining cDNA clones to spot on
macroarray Spotting cDNA clones on macroarray
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How arrays work.
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How arrays work.
cDNA clones spotted on array Obtained
from DD cDNA libraries Subtractive
hybridization Degenerate primers
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How arrays work.
cDNA clones spotted on array Obtained
from DD cDNA libraries Subtractive
hybridization Degenerate primers
TARGETS
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How arrays work.
RNA (total or mRNA) that is labeled
cDNA clones spotted on array Obtained
from DD cDNA libraries Subtractive
hybridization Degenerate primers
TARGETS
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How arrays work.
RNA (total or mRNA) that is labeled
PROBES
cDNA clones spotted on array Obtained
from DD cDNA libraries Subtractive
hybridization Degenerate primers
TARGETS
79
How arrays work.
RNA (total or mRNA) that is labeled
PROBES
cDNA clones spotted on array Obtained
from DD cDNA libraries Subtractive
hybridization Degenerate primers
TARGETS
80
How arrays work.
Matrix Membranes Glass slides Plastic slides
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Matrix (glass slides)
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Matrix (glass slides)
Advantages
-One N per slide. -Can spot thousands of
genes on slides.
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Matrix (glass slides)
Advantages
-One N per slide. -Can spot thousands of
genes on slides.
Disadvantages
-Generally higher background on
slides. -Unequal labeling efficiency.
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Matrix (Membranes)
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Matrix (Membranes)
Advantages
-Low background. -Technology well worked out.
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Matrix (Membranes)
Advantages
-Low background. -Technology well worked out.
Disadvantages
-Two membranes per N. -Can only spot
hundreds of cDNA clones.
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Matrix (Plastic)
?????
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Matrix (Plastic)
Advantages
-2 N per slide.
?????
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Matrix (Plastic)
Advantages
-2 N per slide.
Disadvantages
?????
-Technology is new.
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Spotting cDNA clones on macroarray
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Spotting cDNA clones on macroarray
Vector
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Spotting cDNA clones on macroarray
Vector
PCR amplify
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Spotting cDNA clones on macroarray
Vector
PCR amplify
PCR purify (kit) Concentrate (speedvac) Denature
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Spotting cDNA clones on macroarray
Vector
PCR amplify
PCR purify (kit) Concentrate (speedvac) Denature
Robotically spot 1-10 ng on membrane (UV
crosslink)
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Spotting cDNA clones on macroarray
TARGETS
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Spotting cDNA clones on macroarray
RNA (total or mRNA) that is labeled
Hybridize
TARGETS
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Spotting cDNA clones on macroarray
RNA (total or mRNA) that is labeled
Hybridize Wash
TARGETS
98
Spotting cDNA clones on macroarray
RNA (total or mRNA) that is labeled
Hybridize Wash Detection
TARGETS
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Spotting cDNA clones on macroarray
RNA (total or mRNA) that is labeled
Hybridize Wash Detection Data analysis
TARGETS
100
(No Transcript)
101
Macroarray technology
102
Overview
Lecture One How to obtain cDNA clones to spot on
macroarrays How macroarrays are spotted
Lecture Two Normalization and analysis of
data Demonstration of macroarray spotting
103
Some chemicals found in the environment influence
endocrine pathways (endocrine disruptors) Our
lab is interested in chemicals that disrupt the
estrogen pathway.
104
Estrogen Pathway
Liver
Ovary
Estrogen
Oocyte
ER Receptor
Vtg
Follicle Cells
Adapted from Tata and Smith,1979 Rec Prog Horm Res
105
Some endocrine disrupting chemicals found in the
environment that bind to ER
Ethinylestradiol used in oral
contraceptives. Methoxychlor organochlorine
used in pesticides. Nonylphenol by product of
alkyl phenols, which are
used in surfactants and emulsifiers.
106
Estrogen Pathway
EC
Liver
ER Receptor
Vtg
107
Estrogen Pathway
EC
Liver
ER Receptor
Expression in male and female fish
Vtg
Vtg synthesis and release
108
Develop a macroarray chip which will be used to
monitor the presence and distribution of
estrogenic compounds in coastal habitats.
109
Model system used to generate the targets and
probes were male sheepshead minnows (Cyprinodon
variegatus)
110
Generating targets
54 clones that were spotted on the array
were isolated from differential display (DD)
analysis.
Some clones were E2 up-regulated. Some clones
were E2 down-regulated. Some clones were
constitutive.
111
Various controls were also spotted on the arrays.
Spiking genes (Arabidopsis)
Hybridization efficiency.
112
Spotting cDNA clones on macroarray
RNA (total or mRNA) that is labeled
RNA (total or mRNA) that is labeled
Hybridize
113
Spotting cDNA clones on macroarray
RNA (total or mRNA) that is labeled Plus spiking
RNA
RNA (total or mRNA) that is labeled Plus spiking
RNA
Hybridize
Spiking genes spotted on arrays
114
Spotting cDNA clones on macroarray
RNA (total or mRNA) that is labeled Plus spiking
RNA
RNA (total or mRNA) that is labeled Plus spiking
RNA
Hybridize
Spiking genes spotted on arrays
115
Various controls were also spotted on the arrays.
Spiking genes (Arabidopsis) M13 sequence
(vector, but no cDNA insert)
Hybridization efficiency. How much binding to
vector sequence.
116
pGEM
PCR amplify
Spot on a array
117
pGEM
pGEM
PCR amplify
PCR amplify
Spot on a array
Spot on a array
118
Various controls were also spotted on the arrays.
Spiking genes (Arabidopsis) M13 sequence
(vector, but no cDNA insert) Poly A
stretch
Hybridization efficiency. How much binding to
vector sequence. How much binding to poly
A regions of clones.
119
Clones spotted on array were originally Isolated
from DD.
cDNA(s)
Coding region
5 UTR
3 UTR
AAAAAAAn
UCG
Arbitrary Primers (80)
120
Various controls were also spotted on the arrays.
Spiking genes (Arabidopsis) M13 sequence
(vector, but no cDNA insert) Poly A
stretch Cot-1 repetitive sequences
Hybridization efficiency. How much binding to
vector sequence. How much binding to poly
A regions of clones. Pre-hybridization
efficiency.
121
RNA (total or mRNA) that is labeled
Pre-Hybridize Block with repetitive
sequences (salmon sperm, cot-1)
Hybridize
122
RNA (total or mRNA) that is labeled
Pre-Hybridize Block with repetitive
sequences (salmon sperm, cot-1)
Hybridize
Add ss or cot-1 Sequence to array
123
Generating probes
Radiolabeled total RNA from livers of SHMs
that were exposed to estrogenic
compounds or TEG (control fish).
124
Validation of arrays
How reproducible is one membrane to another?
Aliquots of identical RNA (from C or E2) were
hybridized to two separate membranes.
125
Validation of arrays
Blot 1
Blot 2
126
Normalization of arrays
127
Normalization of arrays
Aliquot of RNA that is labeled
Aliquot of RNA that is labeled
Hybridize
128
Normalization of arrays
Aliquot of RNA that is labeled If slightly more
RNA was labeled
Aliquot of RNA that is labeled
Hybridize
129
Normalization of arrays
Aliquot of RNA that is labeled If slightly more
RNA was labeled
Aliquot of RNA that is labeled
Hybridize
Overall signal (of all the spots) will be
slightly higher
130
Normalization of arrays
Aliquot of RNA that is labeled If slightly more
RNA was labeled
Aliquot of RNA that is labeled
Hybridize
Overall signal (of all the spots) will be
slightly higher
131
Normalization of arrays
Aliquot of RNA that is labeled If slightly more
RNA was labeled
Aliquot of RNA that is labeled
Hybridize
C1 vs. C2 E1 vs. E2
Overall signal (of all the spots) will be
slightly higher
132
Normalization of arrays
Aliquot of RNA that is labeled If slightly more
RNA was labeled
Aliquot of RNA that is labeled
Hybridize
C1 vs. C2 E1 vs. E2 C vs. E
Overall signal (of all the spots) will be
slightly higher
133
Normalization of arrays (if expression pattern of
cDNAs that are spotted are normally distributed)
Spotted high numbers of clones (1000s)
134
Normalization of arrays (if expression pattern of
cDNAs that are spotted are normally distributed)
Spotted high numbers of clones (1000s)
cDNAs
C
E
Expression
135
Normalization of arrays (if expression pattern of
cDNAs that are spotted are normally distributed)
Spotted high numbers of clones (1000s)
cDNAs
C
E
Expression
136
Normalization of arrays (if expression pattern of
cDNAs that are spotted are normally distributed)
Spotted high numbers of clones (1000s)
cDNAs
C
E
Expression
137
Normalization of arrays (if expression pattern of
cDNAs that are spotted are normally distributed)
Spotted high numbers of clones (1000s)
cDNAs
C
E
Expression
138
Normalization of arrays (if expression pattern of
cDNAs that are spotted are normally distributed)
Spotted high numbers of clones (1000s)
Up regulated genes
cDNAs
C
E
Expression
139
Normalization of arrays (clones isolated from DD
are not normally distributed)
Spotted low numbers of clones (100s)
140
Normalization of arrays (clones isolated from DD
are not normally distributed)
Spotted low numbers of clones (100s)
E
C
cDNAs
Expression
141
Normalization of arrays (clones isolated from DD
are not normally distributed)
Spotted low numbers of clones (100s)
E
C
cDNAs
Expression
142
Normalization of arrays (clones isolated from DD
are not normally distributed)
Spotted low numbers of clones (100s)
cDNAs
C
E
Expression
143
Normalization of arrays (clones isolated from DD
are not normally distributed)
Spotted low numbers of clones (100s)
cDNAs
C
E
Expression
144
Normalization of arrays (clones isolated from DD
are not normally distributed)
Spotted low numbers of clones (100s)
Should be up regulated genes
cDNAs
C
E
Expression
145
Normalization of arrays Housekeeping
genes Expression of genes is same for
experimental and control samples (need to
verify).
146
Normalization of arrays Housekeeping
genes Expression of genes is same for
experimental and control samples (need to
verify). -Beta actin -Alpha tubulin -18S
ribosomal -Histones
147
Normalization of arrays Genes found to be
constitutive by chance Identified via DD, cDNA
library etc
148
Constant
Induced
Induced
149
Validation of arrays
Blot 1
Blot 2
150
Validation of arrays
How reproducible is one membrane to another?
Aliquots of identical RNA (from C or E2) were
hybridized to two separate membranes.
151
Validation of arrays
152
Develop arrays to monitor estrogenic compounds
153
Develop arrays to monitor estrogenic compounds
Liver
Aqueously exposed male SHMs to (1) E2 (2)
EE2 (3) Methoxychlor (4) Nonylphenol
ER Receptor
Vtg
154
Develop arrays to monitor estrogenic compounds
Liver
Aqueously exposed male SHMs to (1) E2 (2)
EE2 (3) Methoxychlor (4) Nonylphenol
ER Receptor
Exposed fish
Vtg
Unexposed fish
155
Estradiol arrays
156
Estradiol arrays
Control SHM
157
Estradiol arrays
Control SHM
Estradiol treated SHM
158
1.0E7
Control
E2
1.0E6
1.0E5
Log (intensity)
1.0E4
1.0E3
1
5
10
15
20
25
30
35
40
45
50
55
60
cDNA clones
159
1.0E7
Control
Vtg a
Transferrin
E2
ZP2
1.0E6
Vtg ß
1.0E5
Log (intensity)
1.0E4
1.0E3
1
5
10
15
20
25
30
35
40
45
50
55
60
cDNA clones
160
Fold induction (E2/C)
161
Fold induction (E2/C)
1000
1000
100
100
10
10
2
2


log (intensity)
log (intensity)



cDNA clones



1
1









cDNA
clones
cDNA
clones
0.2
0.2
162
Fold induction (E2/C)
1000
1000
100
100
10
10
2
2
log (intensity)
log (intensity)
cDNA clones









1
1








cDNA
clones
cDNA
clones
0.2
0.2
163
Fold induction (E2/C)
1000
1000
100
100
E2 up-regulated genes
10
10
2
2
log (intensity)
log (intensity)
cDNA clones









1
1








cDNA
clones
cDNA
clones
0.2
0.2
164
Fold induction (E2/C)
Vtg a
1000
1000
ZP2
100
100
Vtg ß
E2 up-regulated genes
10
10
2
2
log (intensity)
log (intensity)
cDNA clones









1
1








cDNA
clones
cDNA
clones
0.2
0.2
165
Fold induction (E2/C)
Vtg a
1000
1000
ZP2
100
100
Vtg ß
E2 up-regulated genes
10
10
2
2
log (intensity)
log (intensity)
cDNA clones









1
1








E2 down-regulated genes
cDNA
clones
cDNA
clones
0.2
0.2
166
Fold induction (E2/C)
Vtg a
1000
1000
ZP2
100
100
Vtg ß
E2 up-regulated genes
10
10
2
2
log (intensity)
log (intensity)
cDNA clones









1
1








E2 down-regulated genes
cDNA
clones
cDNA
clones
Transferrin
0.2
0.2
167
Correlation of array data with differential
display and northern data
Control SHM
E2 SHM
168
Correlation of array data with differential
display and northern data (ZP2)
Control SHM
E2 SHM
169
Correlation of array data with differential
display and northern data (ZP2)
Control SHM
E2 SHM
ZP2 (26)
Control
E2
170
Correlation of array data with differential
display and northern data (ZP2)
Control SHM
E2 SHM
ZP2 (26)
Control
E2
Northern
Control
E2
171
Correlation of array data with differential
display and northern data (Vtg a)
Control SHM
E2 SHM
172
Correlation of array data with differential
display and northern data (Vtg a)
Control SHM
E2 SHM
Vtg a (26)
Control
E2
173
Correlation of array data with differential
display and northern data (Vtg a)
Control SHM
E2 SHM
Vtg a (26)
Control
E2
Northern
Control
E2
174
Ethinyl estradiol array Methoxychlor
array Nonylphenol array
175
Control
Estradiol
176
Control
Estradiol
EE2
177
Control
Estradiol
EE2
Methoxychlor
178
Control
Estradiol
EE2
Methoxychlor
Nonylphenol