Introduction to Microarrays.

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Introduction to Microarrays

The Central Dogma

protein DNA RNA Life - a recipe for making proteins Transcription Translation DNA RNA

ATCTTTTTCGGCTTTTTTTAGTATCCACAGAGGTTATCGACAACATTTTCACATTACCAACCCCTGTGGACAAGGTTTTTTCAACAGGTTGTCCGCTTTGTGGATAAGATTGTGACAACCATTGCAAGCTCTCGTTTATTTTGGTATTATATTTGTGTTTTAACTCTTGATTACTAATCCTACCTTTCCTCTTTATCCACAAAGTGTGGATAAGTTGTGGATTGATTTCACACAGCTTGTGTAGAAGGTTGTCCACAAGTTGTGAAATTTGTCGAAAAGCTATTTATCTACTATATTATATGTTTTCAACATTTAATGTGTACGAATGGTAAGCGCCATTTGCTCTTTTTTTGTGTTCTATAACAGAGAAAGACGCCATTTTCTAAGAAAAGGAGGGACGTGCCGGAAGATGGAAAATATATTAGACCTGTGGAACCAAGCCCTTGCTCAAATCGAAAAAAAGTTGAGCAAACCGAGTTTTGAGACTTGGATGAAGTCAACCAAAGCCCACTCACTGCAAGGCGATACATTAACAATCACGGCTCCCAATGAATTTGCCAGAGACTGGCTGGAGTCCAGATACTTGCATCTGATTGCAGATACTATATATGAATTAACCGGGGAAGAATTGAGCATTAAGTTTGTCATTCCTCAAAATCAAGATGTTGAGGACTTTATGCCGAAACCGCAAGTCAAAAAAGCGGTCAAAGAAGATACATCTGATTTTCCTCAAAATATGCTCAATCCAAAATATACTTTTGATACTTTTGTCATCGGATCTGGAAACCGATTTGCACATGCTGCTTCCCTCGCAGTAGCGGAAGCGCCCGCGAAAGCTTACAACCCTTTATTTATCTATGGGGGCGTCGGCTTAGGGAAAACACACTTAATGCATGCGATCGGCCATTATGTAATAGATCATAATCCTTCTGCCAAAGTGGTTTATCTGTCTTCTGAGAAATTTACAAACGAATTCATCAACTCTATCCGAGATAATAAAGCCGTCGACTTCCGCAATCGCTATCGAAATGTTGATGTGCTTTTGATAGATGATATTCAATTTTTAGCGGGGAAAGAACAAACCCAGGAAGAATTTTTCCATACATTTAACACATTACACGAAGAAAGCAAACAAATCGTCATTTCAAGTGACCGGCCGCCAAAGGAAATTCCGACACTTGAAGACAGATTGCGCTCACGTTTTGAATGGGGACTTATTACAGATATCACACCGCCTGATCTAGAAACGAGAATTGCAATTTTAAGAAAAAAGGCCAAAGCAGAGGGCCTCGATATTCCGAACGAGGTTATGCTTTACATCGCGAATCAAATCGACAGCAATATTCGGGAACTCGAAGGAGCATTAATCAGAGTTGTCGCTTATTCATCTTTAATTAATAAAGATATTAATGCTGATCTGGCCGCTGAGGCGTTGAAAGATATTATTCCTTCCTCAAAACCGAAAGTCATTACGATAAAAGAAATTCAGAGGGTAGTAGGCCAGCAATTTAATATTAAACTCGAGGATTTCAAAGCAAAAAAACGGACAAAGTCAGTAGCTTTTCCGCGTCAAATCGCCATGTACTTATCAAGGGAAATGACTGATTCCTCTCTTCCTAAAATCGGTGAAGAGTTTGGAGGACGTGATCATACGACCGTTATTCATGCGCATGAAAAAATTTCAAAACTGCTGGCAGATGATGAACAGCTTCAGCAGCATGTAAAAGAAATTAAAGAACAGCTTAAATAGCAGGACCGGGGATCAATCGGGGAAAGTGTGAATAACTTTTCGGAAGTCATACACAGTCTGTCCACATGTGGATAGGCTGTGTTTCCTGTCTTTTTCACAACTTATCCACAAATCCACAGGCCCTACTATTACTTCTACTATTTTTTATAAATATATATATTAATACATTATCCGTTAGGAGGATAAAAATGAAATTCACGATTCAAAAAGATCGTCTTGTTGAAAGTGTCCAAGATGTATTAAAAGCAGTTTCATCCAGAACCACGATTCCCATTCTGACTGGTATTAAAATTGTTGCATCAGATGATGGAGTATCCTTTACAGGGAGTGACTCAGATATTTCTATTGAATCCTTCATTCCAAAAGAAGAAGGAGATAAAGAAATCGTCACTATTGAACAGCCCGGAAGCATCGTTTTACAGGCTCGCTTTTTTAGTGAAATTGTAAAAAAATTGCCGATGGCAACTGTAGAAATTGAAGTCCAAAATCAGTATTTGACGATTATCCGTTCTGGTAAAGCTGAATTTAATCTAAACGGACTGGATGCTGATGAATATCCGCACTTGCCGCAGATTGAAGAGCATCATGCGATTCAGATCCCAACTGATTTGTTAAAAAATCTAATCAGACAAACAGTATTTGCAGTGTCCACCTCAGAAACACGCCCTATCTTGACAGGTGTAAACTGGAAAGTGGAGCAAAGTGAATTATTATGCACTGCAACGGATAGCCACCGTCTTGCATTAAGAAAGGCGAAACTTGATATTCCAGAAGACAGATCTTATAACGTCGTGATTCCGGGAAAAAGTTTAACTGAACTCAGCAAGATTTTAGATGACAACCAGGAACTTGTAGATATCGTCATCACAGAAACCCAAGTTCTGTTTAAAGCGAAAAACGTCTTGTTCTTCTCACGGCTTCTGGACGGGAATTATCCAGACACAACCAGCCTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACAAATTGCCGATGGCAACTGTAGAAATTGAAGTCCAAAATCAGTATTTGACGATTATCCGTTCTGGTAAAGCTGAATTTAATCTAAACGGACTGGATGCTGATGAATATCCGCACTTGCCGCAGATTGAAGAGCATCATGCGATTCAGATCCCAACTGATTTGTTAAAAAATCTAATCAGACAAACAGTATTTGCAGTGTCCACCTCAGAAACACGCCCTATCTTGACAGGTGTAAACTGGAAAGTGGAGCAAAGTGAATTATTATGCACTGCAACGGATAGCCACCGTCTTGCATTAAGAAAGGCGAAACTTGATATTCCAGAAGACAGATCTTATAACGTCGTGATTCCGGGAAAAAGTTTAACTGAACTCAGCAAGATTTTAGATGACAACCAGGAACTTGTAGATATCGTCATCACAGAAACCCAAGTTCTGTTTAAAGCGAAAAACGTCTTGTTCTTCTCACGGCTTCTGGACGGGAATTATCCAGACACAACCAGCCTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACACAGACACAACCAGCCTGATTCCGCAAGACAGCAAAACAGAAATCATTGTGAACACAAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACAAAGAATTCCTTCAGGCCATTGATCGTGCATCTCTTTTAGCTAGAGAGGGACGCAACATTGTGA DNA

The Central Dogma

Hybridization A T G C T A G C A T G C

Introduction to Microarrays

Microarrays - The Concept Measure the level of transcript from a very large number of genes in one go RNA CELL

Microarrays - The Technologies Stanford Microarrays High-density

Why? RNA

gene specific DNA probes How? gene mRNA gene specific DNA probes labeled target

Stanford-type Microarrays

Stanford-type Microarrays Coating glass slides Deposition of probes Post-processing Hybridization

Making Microarrays 1. Produce probes 2. Print by the use of a robot oligos cDNA library PCR products 1. Produce probes 2. Print by the use of a robot

Spotting - Mechanical deposition of probes

16-pin microarrayer

Microarrayer

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

Sample preparation 1. Design experiment 2. Perform experiment wild type mutant 1. Design experiment Question? Replicates? Test? 2. Perform experiment 3. Precipitate RNA Eukaryote/prokaryote? Cell wall? 4. Label RNA Amplification? Direct or indirect? Label?

Stanford microarrays DESIGN and ORDER PROBES SAMPLE CONTROL mRNA cDNA Cy3-cDNA Cy5-cDNA

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

Catalog Arrays Human E. coli Mouse P. aeruginosa Rat Arabidopsis C. elegans Canine Drosophila E. coli P. aeruginosa Plasmodium/Anopheles Vitis vinifera (Grape) Xenopus laevis Yeast Zebrafish NimbleExpress™ Array Program

Fluidic Station and Scanner

The Affymetrix Genechip®

Photolithography Mask #2 Mask #1 in situ synthesis T A Mask #2 Mask #1 T A Spacers bound to surface with photolabile protection groups

Photolithography - Micromirrors NimbleExpress™ Array Program manufactured on Iceland by NimbleGen Systems Inc.

The Affymetrix GeneChip® A gene is represented like this: PM MM - Perfect Match (PM) - MisMatch (MM) PM: CGATCAATTGCACTATGTCATTTCT MM: CGATCAATTGCAGTATGTCATTTCT

NimbleGen Systems Inc. ~380.000 probes/array They do most of the practical work

The Technologies - Flexibility Stanford microarrays: Are flexible, but new probes must be ordered each time High-density: Are not flexible, ....unless you order the NimbleExpress™ chip or use the NimbleGen Systems

Analysis of Data Normalization: Linear or non-linear

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

Analysis of Data Normalization: Linear or non-linear Statistical test: student’s t-test ANalysis Of VAriance (ANOVA) Analysis: Principle Component Analysis (PCA) Clustering and visualization