Introduction to

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Introduction to Microarrays
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The Central Dogma
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Life - a recipe for making proteins
Translation
Transcription
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ATCTTTTTCGGCTTTTTTTAGTATCCACAGAGGTTATCGACAACATTTTC
ACATTACCAACCCCTGTGGACAAGGTTTTTTCAACAGGTTGTCCGCTTTG
TGGATAAGATTGTGACAACCATTGCAAGCTCTCGTTTATTTTGGTATTAT
ATTTGTGTTTTAACTCTTGATTACTAATCCTACCTTTCCTCTTTATCCAC
AAAGTGTGGATAAGTTGTGGATTGATTTCACACAGCTTGTGTAGAAGGTT
GTCCACAAGTTGTGAAATTTGTCGAAAAGCTATTTATCTACTATATTATA
TGTTTTCAACATTTAATGTGTACGAATGGTAAGCGCCATTTGCTCTTTTT
TTGTGTTCTATAACAGAGAAAGACGCCATTTTCTAAGAAAAGGAGGGACG
TGCCGGAAGATGGAAAATATATTAGACCTGTGGAACCAAGCCCTTGCTCA
AATCGAAAAAAAGTTGAGCAAACCGAGTTTTGAGACTTGGATGAAGTCAA
CCAAAGCCCACTCACTGCAAGGCGATACATTAACAATCACGGCTCCCAAT
GAATTTGCCAGAGACTGGCTGGAGTCCAGATACTTGCATCTGATTGCAGA
TACTATATATGAATTAACCGGGGAAGAATTGAGCATTAAGTTTGTCATTC
CTCAAAATCAAGATGTTGAGGACTTTATGCCGAAACCGCAAGTCAAAAAA
GCGGTCAAAGAAGATACATCTGATTTTCCTCAAAATATGCTCAATCCAAA
ATATACTTTTGATACTTTTGTCATCGGATCTGGAAACCGATTTGCACATG
CTGCTTCCCTCGCAGTAGCGGAAGCGCCCGCGAAAGCTTACAACCCTTTA
TTTATCTATGGGGGCGTCGGCTTAGGGAAAACACACTTAATGCATGCGAT
CGGCCATTATGTAATAGATCATAATCCTTCTGCCAAAGTGGTTTATCTGT
CTTCTGAGAAATTTACAAACGAATTCATCAACTCTATCCGAGATAATAAA
GCCGTCGACTTCCGCAATCGCTATCGAAATGTTGATGTGCTTTTGATAGA
TGATATTCAATTTTTAGCGGGGAAAGAACAAACCCAGGAAGAATTTTTCC
ATACATTTAACACATTACACGAAGAAAGCAAACAAATCGTCATTTCAAGT
GACCGGCCGCCAAAGGAAATTCCGACACTTGAAGACAGATTGCGCTCACG
TTTTGAATGGGGACTTATTACAGATATCACACCGCCTGATCTAGAAACGA
GAATTGCAATTTTAAGAAAAAAGGCCAAAGCAGAGGGCCTCGATATTCCG
AACGAGGTTATGCTTTACATCGCGAATCAAATCGACAGCAATATTCGGGA
ACTCGAAGGAGCATTAATCAGAGTTGTCGCTTATTCATCTTTAATTAATA
AAGATATTAATGCTGATCTGGCCGCTGAGGCGTTGAAAGATATTATTCCT
TCCTCAAAACCGAAAGTCATTACGATAAAAGAAATTCAGAGGGTAGTAGG
CCAGCAATTTAATATTAAACTCGAGGATTTCAAAGCAAAAAAACGGACAA
AGTCAGTAGCTTTTCCGCGTCAAATCGCCATGTACTTATCAAGGGAAATG
ACTGATTCCTCTCTTCCTAAAATCGGTGAAGAGTTTGGAGGACGTGATCA
TACGACCGTTATTCATGCGCATGAAAAAATTTCAAAACTGCTGGCAGATG
ATGAACAGCTTCAGCAGCATGTAAAAGAAATTAAAGAACAGCTTAAATAG
CAGGACCGGGGATCAATCGGGGAAAGTGTGAATAACTTTTCGGAAGTCAT
ACACAGTCTGTCCACATGTGGATAGGCTGTGTTTCCTGTCTTTTTCACAA
CTTATCCACAAATCCACAGGCCCTACTATTACTTCTACTATTTTTTATAA
ATATATATATTAATACATTATCCGTTAGGAGGATAAAAATGAAATTCACG
ATTCAAAAAGATCGTCTTGTTGAAAGTGTCCAAGATGTATTAAAAGCAGT
TTCATCCAGAACCACGATTCCCATTCTGACTGGTATTAAAATTGTTGCAT
CAGATGATGGAGTATCCTTTACAGGGAGTGACTCAGATATTTCTATTGAA
TCCTTCATTCCAAAAGAAGAAGGAGATAAAGAAATCGTCACTATTGAACA
GCCCGGAAGCATCGTTTTACAGGCTCGCTTTTTTAGTGAAATTGTAAAAA
AATTGCCGATGGCAACTGTAGAAATTGAAGTCCAAAATCAGTATTTGACG
ATTATCCGTTCTGGTAAAGCTGAATTTAATCTAAACGGACTGGATGCTGA
TGAATATCCGCACTTGCCGCAGATTGAAGAGCATCATGCGATTCAGATCC
CAACTGATTTGTTAAAAAATCTAATCAGACAAACAGTATTTGCAGTGTCC
ACCTCAGAAACACGCCCTATCTTGACAGGTGTAAACTGGAAAGTGGAGCA
AAGTGAATTATTATGCACTGCAACGGATAGCCACCGTCTTGCATTAAGAA
AGGCGAAACTTGATATTCCAGAAGACAGATCTTATAACGTCGTGATTCCG
GGAAAAAGTTTAACTGAACTCAGCAAGATTTTAGATGACAACCAGGAACT
TGTAGATATCGTCATCACAGAAACCCAAGTTCTGTTTAAAGCGAAAAACG
TCTTGTTCTTCTCACGGCTTCTGGACGGGAATTATCCAGACACAACCAGC
CTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATT
CCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACA
AATTGCCGATGGCAACTGTAGAAATTGAAGTCCAAAATCAGTATTTGACG
ATTATCCGTTCTGGTAAAGCTGAATTTAATCTAAACGGACTGGATGCTGA
TGAATATCCGCACTTGCCGCAGATTGAAGAGCATCATGCGATTCAGATCC
CAACTGATTTGTTAAAAAATCTAATCAGACAAACAGTATTTGCAGTGTCC
ACCTCAGAAACACGCCCTATCTTGACAGGTGTAAACTGGAAAGTGGAGCA
AAGTGAATTATTATGCACTGCAACGGATAGCCACCGTCTTGCATTAAGAA
AGGCGAAACTTGATATTCCAGAAGACAGATCTTATAACGTCGTGATTCCG
GGAAAAAGTTTAACTGAACTCAGCAAGATTTTAGATGACAACCAGGAACT
TGTAGATATCGTCATCACAGAAACCCAAGTTCTGTTTAAAGCGAAAAACG
TCTTGTTCTTCTCACGGCTTCTGGACGGGAATTATCCAGACACAACCAGC
CTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATT
CCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACA
CAGACACAACCAGCCTGATTCCGCAAGACAGCAAAACAGAAATCATTGTG
AACACAAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAG
AGAGGGACGCAACAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTT
TAGCTAGAGAGGGACGCAACATTGTGA
DNA
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The Central Dogma
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Hybridization
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Introduction to Microarrays
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Microarrays - The Concept
Measure the level of transcript from a very large
number of genes in one go
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Microarrays - The Technologies
Stanford Microarrays High-density
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Why?
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How?
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Stanford-type Microarrays
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Stanford-type Microarrays
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Making Microarrays

• oligos
• cDNA library
• PCR products

1. Produce probes
2. Print by the use of a robot
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Spotting - Mechanical deposition of probes
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16-pin microarrayer
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(No Transcript)
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Microarrayer
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Making Microarrays

• oligos
• cDNA library
• PCR products

1. Produce probes
2. Print by the use of a robot

• 3. Post-process
• rehydrate
• snap dry
• UV-cross link
• block surface

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Sample preparation
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Stanford microarrays
DESIGN and ORDER PROBES
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Affymetrix GeneChip oligonucleotide array

• 11 to 20 oligonucleotide probes for each gene
• On-chip synthesis of 25 mers
• 20.000 genes per chip
• good quality data low variance

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Catalog Arrays
NimbleExpress Array Program
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Fluidic Station and Scanner
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The Affymetrix Genechip
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Photolithography
in situ synthesis
Spacers bound to surface with photolabile
protection groups
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Photolithography - Micromirrors
NimbleExpress Array Program manufactured on
Iceland by NimbleGen Systems Inc.
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The Affymetrix GeneChip

• A gene is represented like this

- Perfect Match (PM) - MisMatch (MM)
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NimbleGen Systems Inc.
380.000 probes/array They do most of the
practical work
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The Technologies - Flexibility
Stanford microarrays Are flexible, but new
probes must be ordered each timeHigh-density
Are not flexible, ....unless you order the
NimbleExpress chip or use the NimbleGen Systems
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Analysis of Data

• Normalization
• Linear or non-linear

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Is it worth it?
Known positives versus the total number of
significantly affected genes at 5 different
cutoffs in the TnrA experiment
Number of known positives
Qspline normalization Linear normalization
Number of significantly affected genes
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Analysis of Data

• Normalization
• Linear or non-linear

Statistical test students t-test ANalysis Of
VAriance (ANOVA)
Analysis Principle Component Analysis (PCA)
Clustering and visualization