What is a DNA Microarray? - Massachusetts Institute of...
Transcript of What is a DNA Microarray? - Massachusetts Institute of...
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An Introduction to DNA microarrays
Rebecca Fry, Ph.D.
http://www.buffalo.edu/UBT/UBT
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 oligonucleotidearrays”
Schena et al., 1995 Science“Quantitative monitoring of gene expression patterns with a complementary DNA microarray”
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
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 intoamino acids that make up proteins
Process: “TRANSLATION”
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
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 microarraysIntroduction
BRCA2
BRCA1
BLM
BID
ATR
ATM
APEXL2
APEX
ADPRTL3
ADPRTL2
ADPRT
ABH3
ABH2
ABH
DDB1
DCLRE1A
CETN2
CDK8
CDK7
CDK6
CDK4
CDK2
CCNH
CASP9
CASP8
CASP3
CASP2
BTG1
EXO1
FANCA
FANCC
FANCE
FANCF
ERCC5
ERCC4
ERCC3
ERCC2
ERCC1
ENDOG
EGFP
DUT
DMC1
MAD2L2
LIG4
LIG3
LIG1
JUN
HUS1
HSU24186
HCNP
HAP1
GTF2H4
GADD45G
GADD45B
GADD45A
G22P1 POLQ
PRKDC
PRSS25
RAD1
RAD17
RAD18
RAD23A
RAD23B
RAD50
RAD51
RAD51C
RAD51L1
RAD51L3
RAD52
RAD54B
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
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
Introductioncy3 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=cy5red cy5>cy3
green cy3>cy5
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 kb0.5 kb0.2 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!!
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
Information:
Gene nameOligo idCy3 intensityCy5 intensity
Image Analysis: Spotted arraysWhat information do we see?
117.8919272.5676H200008289ADPRT
108.9855259.5362H200008289ADPRT
108.5797265.7391H200008289ADPRT
131.2286324.886H200008289ADPRT
115.6119296.5224H200014101PRKDC
117.8676306.8235H200014101PRKDC
129.5333340.933H200014101PRKDC
132348.522H200014101PRKDC
2351.6976242.5control1control1
2370.5756434.93control1control1
2068.515786.72control1control1
2371.1636642.5control1control1
Spot Mean Intensity (w685)
Spot Mean Intensity (w595)Clone IDName
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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S c a tte r P lo t
dChip e xpre s s iontis s 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
EGFP
ver
sus
EGFP
KD
Rat
io L
og2
Data AnalysisRequires software: Spotfire
Requires ability to search for Patterns and Trends