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An Introduction to DNA microarrays
Rebecca Fry, Ph.D. (and Leona Samson) The title of this talk is the design and implementation fo t DNA microarray experiments
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What is a DNA Microarray?
genes or gene fragments attached to a substrate (glass) Tens of thousands of spots/genes =entire genome in 1 experiment A Revolution in Biology Hybridized slide Two dyes Image analyzed
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mRNA Analysis Methods Northern Blot (Single Gene analysis)
Microarray Technology (Genome Wide Experiment)
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NORTHERN BLOTS mRNAs separated on gel according to size
mRNAs transferred to a membrane and hybridized with small number (1-5) of radioactively labeled DNA probes. Probe corresponds to gene of interest Target RNA is spatially fixed and the labeled probe is in solution Low throughput
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i.e., your cloned gene(s)
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Number of Genes in Different Organisms
Yeast ~ 6200 genes E. coli ~ 4200 genes Human ~ 30,000 genes Mouse ~ 30,000 genes Phage T4 ~ 200 genes Influenza ~ 12 genes
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We could just 35,000 Northern to monitor expression of all genes!!!
i.e., your cloned gene(s) We could just 35,000 Northern to monitor expression of all genes!!!
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Northern Blots DNA Microarrays
Immobilized mRNA population hybridized with labeled probe representing one gene DNA Microarrays Immobilized probes hybridized with labeled mRNA population representing all expressed genes
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Need to achieve two things:
Immobilize thousands of probes specific for individual genes Label mRNA populations
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Designing Oligo Probes
1 846 EGFP ORF ATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGCGGTGGCGGCGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAGGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGA 70 mer oligo specific to gene of interest
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Number of Genes in Different Organisms
Yeast ~ 6200 genes E. coli ~ 4200 genes Human ~ 30,000 genes Mouse ~ 30,000 genes Phage T4 ~ 200 genes Influenza ~ 12 genes
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Overview of fabrication of spotted microarrays Resuspension of oligos
Introduction Overview of fabrication of spotted microarrays Liquid Handling Resuspension of oligos Spotted microarrays Robotic Printing
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Need to achieve two things:
Immobilize thousands of probes specific for individual genes Label mRNA populations
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cy3 and cy5: Commonly used dyes
Introduction cy3 and cy5: Commonly used dyes cy5 emission 664 nm cy3 cy5 Differential dye incorporation cy5 less well than cy3 Light sensitivity: cy5 more easily degraded Protect your reactions from light!! Always difference between cy3 and cy5, there is obvious structural difference with an additional double bond 562 nm emission 510 nm cy3
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Reverse transcription
Introduction Spotted microarray target preparation Direct labeling Target preparation Labeled cDNA preparation Spotted Microarray EGFP Sample 2 EGFP KD RNA cy3 Reverse transcription Flourescent dyes cy5 cDNA cDNA Combined in equal amounts Co-hybridized to array
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Reverse transcription
Introduction Spotted microarray target preparation Direct labeling Target preparation Labeled cDNA preparation Spotted Microarray EGFP Sample 2 EGFP KD RNA cy3 Reverse transcription Flourescent dyes cy5 cDNA cDNA Combined in equal amounts yellow cy3=cy5 red cy5>cy3 green cy3>cy5 Co-hybridized to array
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cDNA “Two Color Chips” summary Target preparation Array preparation
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This is the kind of thing you will see in YOUR microarray experiments
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What’s happening at each spot?
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cDNA Chip vs. Northern Blot
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Commercially available
Introduction Two Popular Microarraying Platforms Spotted microarrays Commercially available Oligo microarray cDNA: PCR products (500-1,000bp) synthesized oligos (70 mer) >10,000 probes Affymetrix “Gene Chip” 500,000 probes 25 mer (represents a fragment of a gene)
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So..what gene probes are represented on the array you will use??
EGFP, p53, EXO1, AAG, ATM, and ATR…. But we added in a bunch more!!
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Alien DNA No homology to Human sequences
Growth arrest and DNA-damage-inducible POLQ PRKDC PRSS25 RAD1 RAD17 RAD18 RAD23A RAD23B RAD50 RAD51 RAD51C RAD51L1 RAD51L3 RAD52 RAD54B RAD54L RAD9 RECQL4 REV1L ABH ABH2 ABH3 ADPRT ADPRTL2 ADPRTL3 APEX APEXL2 ATM ATR BID BLM BRCA1 BRCA2 alkB homolog BTG1 CASP2 CASP3 CASP8 CASP9 CCNH CDK2 CDK4 CDK6 CDK7 CDK8 CETN2 DCLRE1A DDB1 DDB2 G22P1 GADD45A GADD45B GADD45G GTF2H4 HAP1 HCNP HSU24186 HUS1 JUN LIG1 LIG3 LIG4 MAD2L2 DMC1 DUT EGFP ENDOG ERCC1 ERCC2 ERCC3 ERCC4 ERCC5 EXO1 FANCA FANCC FANCE FANCF FEN1 FOS ERCC6 No homology to Human sequences Alien DNA Excision repair Breast cancer 1
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Electropherogram (28S/18S Ratio~2)
RNA quality control Pre-labeling quality control: Determine RNA Quality Agilent Bioanalyzer: ng No more formaldehyde gels!! Gel Image (in silico) Sharp, Clear Bands Liver Electropherogram (28S/18S Ratio~2) 6 kb 4 kb 28S 28S 2 kb 18S 1 kb 0.5 kb 0.2 kb Lad, 1,2
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Image Analysis: Spotted arrays
Microarray Measurements Image Analysis: Spotted arrays Image Analysis Scanner .txt or .xls file
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