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An Introduction to DNA microarrays
Rebecca Fry, Ph.D.
http://www.buffalo.edu/UBT/UBT
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What is a DNA Microarray?
genes or gene fragments attached to a substrate (glass)
Hybridized slide
Two dyes
Image analyzed
Tens of thousands of spots
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The Beginnings of Microarray Technology
Lockhart et al., 1996Nature Biotechnology“Expression monitoring by hybridisation to high-density oligonucleotide arrays”
Schena et al., 1995 Science“Quantitative monitoring of gene expression patterns with a complementary DNA microarray”
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8 years later4162 references
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Uses and Applications
Disease characterizationGene screening
Pathway mapping Target identification
Developmental BiologyPersonalized medicine
Mechanism of Action Studies Molecular Diagnosis of Disease
Toxicology Prediction of Drug Efficacy/Toxicity
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A model experiment: Two samples of interest
Q: Which genes are differentially expressed in EGFP cells versus EGFP KD cells?
EGFP KDEGFP expressing cells
http://www.oardc.ohio-state.edu/plantranslab/gfp-a.gif
Differential
Gene
Expression?!
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Cell
nucleus
cytoplasm
DNA
Inside every cell: DNA Serves as a genetic blueprint
RNA
Protein
Chem.ox.ac.uk
Dna molecule: berkeley.edu
Process of transcribing deoxyribonucleic acid toribonucleic acid is“TRANSCRIPTION”
Nucleic acidsmust be translated into amino acids that make up proteins
Process: “TRANSLATION”
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RNA
Protein
DNA
Relating Gene Expression
Is a gene expressed? Is protein produced
Ideally measure protein levels
High throughput protein assays complicated
We measure transcript level
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Two Popular Microarraying Platforms
www.molgen.mpg.de
Spotted microarrays
cDNA: PCR products (500-2,000bp)synthesized oligos>10,000 probes
Affymetrix“Gene Chip”500,000 probes25 mer (represents a fragment of a gene)
Introduction
Commercially available microarray
www.the-scientist.com
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EGFP ORF
1 846
ATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGCGGTGGCGGCGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAGGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGA
Designing Oligos
70 mer oligo
specific to gene
of interest
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Spotted microarrays
www.biorobotics.com
Robotic Printing
Robotics: Liquid HandlingResuspension of oligos
www.qiageninstruments.com
Overview of fabrication of spotted microarrays
Introduction
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ABH
ABH2
ABH3
ADPRT
ADPRTL2
ADPRTL3
APEX
APEXL2
ATM
ATR
BID
BLM
BRCA1
BRCA2
BTG1
CASP2
CASP3
CASP8
CASP9
CCNH
CDK2
CDK4
CDK6
CDK7
CDK8
CETN2
DCLRE1A
DDB1
DDB2
DMC1
DUT
EGFP
ENDOG
ERCC1
ERCC2
ERCC3
ERCC4
ERCC5
EXO1
FANCA
FANCC
FANCE
FANCF
FEN1
FOS
ERCC6
G22P1
GADD45A
GADD45B
GADD45G
GTF2H4
HAP1
HCNP
HSU24186
HUS1
JUN
LIG1
LIG3
LIG4
MAD2L2
POLQ
PRKDC
PRSS25
RAD1
RAD17
RAD18
RAD23A
RAD23B
RAD50
RAD51
RAD51C
RAD51L1
RAD51L3
RAD52
RAD54B
RAD54L
RAD9
RECQL4
REV1L
alkB homolog
Breast cancer 1
Excision repair
Growth arrest and DNA-damage-inducible
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A closer look at Spotted microarraysSome nomenclature
www.molgen.mpg.de
each spotrepresents a gene or gene fragment
gene
RNA
“probe”
“target”
Introduction
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562 nm
cy3
cy5
www.amersham.com
cy3 cy5
Differential dye incorporationcy5 less well than cy3Light sensitivity: cy5 more easily degraded
664 nm
510 nm
emission
emission
Introduction
cy3 and cy5: Commonly used dyes
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www.genetics.ucla.edu
Labeled cDNA preparation
RNA
Sample 2
Spotted MicroarrayTarget preparation
Reverse transcriptionFlourescent dyes
Introduction
EGFP KD
Combined in equal amounts
Spotted microarray target preparationDirect labeling
cy5cy3
cDNAcDNA
EGFP
Co-hybridized to array
yellow cy3=cy5yellow cy3=cy5
red cy5>cy3red cy5>cy3
green cy3>cy5green cy3>cy5
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DNA
Direct Labeling (Spotted Arrays)
RNA
RT
Reverse transcriptionRNA
Enzyme (Superscript RT)Dye
Oligo d(T)nucleotides
AAAAAAAAA AAA
AAAAAA
EGFP KDEGFP
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28S
Liver
18S
28S
6 kb
4 kb
2 kb
1 kb
0.5 kb
0.2 kb
Electropherogram (28S/18S Ratio~2)
18S
28S
Fluo
resc
ence
Time (seconds)
0
10
20
30
40
50
60
70
19 24 29 34 39 44 49 54 59 64 69
Sample 1 Ladder Sample 2
RNA quality control
Lad, L, P
Gel Image (in silico) Sharp, Clear Bands
Pre-labeling quality control:
Determine RNA Quality Agilent Bioanalyzer: 50-500 ngNo more formaldehyde gels!!
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Microarray Measurements
Scanner Image Analysis .txt or .xls file
Image Analysis: Spotted arrays
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Spotted microarrays
www.molgen.mpg.de
Signal is average of pixel intensities of spot
2 numbers per spot
Microarray Measurements
cy5
cy3
Signal: Spotted arrays
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Information:
Gene nameOligo idCy3 intensityCy5 intensity
Image Analysis: Spotted arraysWhat information do we see?
Name Clone ID
Spot Mean Intensity (w595)
Spot Mean Intensity (w685)
control1 control1 6642.5 2371.163
control1 control1 5786.72 2068.51
control1 control1 6434.93 2370.575
control1 control1 6242.5 2351.697
PRKDCH20001410
1 348.522 132
PRKDCH20001410
1 340.933 129.5333
PRKDCH20001410
1 306.8235 117.8676
PRKDCH20001410
1 296.5224 115.6119
ADPRTH20000828
9 324.886 131.2286
ADPRTH20000828
9 265.7391 108.5797
ADPRTH20000828
9 259.5362 108.9855
ADPRTH20000828
9 272.5676 117.8919
Steps:Normalize globally
Calculate average of cy3 and cy5Bring cy5 numbers to cy3 by multiplying by common factor
Ratios (take ratio of wt/KD…look for 2 fold cutoff)Log2 (calculate log 2 of ratio to differentiate increase or decrease)Reproducibility (how did the four replicates perform?)
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Scatter Plot
dChip expressiontiss diff_ID0 2000 4000 6000 8000 10000 12000
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
7
8
9
2 fold increase
2 fold decrease
Gene population
Plots
EG
FP
ver
sus
EG
FP
KD
R
atio
Log
2
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Data AnalysisRequires software: Spotfire
Requires ability to search for Patterns and Trends
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mammalian DNA damage response pathway
The DNA damage response: puttingcheckpoints in perspectiveBin-Bing S. Zhou* & Stephen J. Elledge²
NATURE |VOL 408 | 23 NOVEMBER 2000 |
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Good luck!