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An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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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). Tens of thousands of spots. Hybridized slide Two dyes Image analyzed. The Beginnings of Microarray Technology. - PowerPoint PPT Presentation
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n Introduction to DNA microarrays Rebecca Fry, Ph.D. http://www.buffalo.edu/UBT/UBT
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Page 1: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

An Introduction to DNA microarrays

Rebecca Fry, Ph.D.

http://www.buffalo.edu/UBT/UBT

Page 2: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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

Page 3: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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”

Page 4: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

8 years later4162 references

Page 5: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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

Page 6: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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?!

Page 7: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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”

Page 8: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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

Page 9: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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

Page 10: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

EGFP ORF

1 846

ATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGCGGTGGCGGCGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAGGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGA

Designing Oligos

70 mer oligo

specific to gene

of interest

Page 11: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

Spotted microarrays

www.biorobotics.com

Robotic Printing

Robotics: Liquid HandlingResuspension of oligos

www.qiageninstruments.com

Overview of fabrication of spotted microarrays

Introduction

Page 12: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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

Page 13: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

A closer look at Spotted microarraysSome nomenclature

www.molgen.mpg.de

each spotrepresents a gene or gene fragment

gene

RNA

“probe”

“target”

Introduction

Page 14: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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

Page 15: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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

Page 16: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

DNA

Direct Labeling (Spotted Arrays)

RNA

RT

Reverse transcriptionRNA

Enzyme (Superscript RT)Dye

Oligo d(T)nucleotides

AAAAAAAAA AAA

AAAAAA

EGFP KDEGFP

Page 17: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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!!

Page 18: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

Microarray Measurements

Scanner Image Analysis .txt or .xls file

Image Analysis: Spotted arrays

Page 19: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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

Page 20: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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?)

Page 21: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

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

Page 22: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

Data AnalysisRequires software: Spotfire

Requires ability to search for Patterns and Trends

Page 23: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

mammalian DNA damage response pathway

The DNA damage response: puttingcheckpoints in perspectiveBin-Bing S. Zhou* & Stephen J. Elledge²

NATURE |VOL 408 | 23 NOVEMBER 2000 |

Page 24: An Introduction to DNA microarrays Rebecca Fry, Ph.D.

Good luck!


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