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Microarray Technologies

Microarray Technologies. Mark Reimers. Outline. Library preparation Hybridization cDNA expression arrays Oligo expression arrays Agilent Affymetrix Illumina NimbleGen Other array types. Preparing a cDNA Library from mRNA. Hybridization. Glass Slide Microarrays (1994).

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Microarray Technologies

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  1. Microarray Technologies Mark Reimers

  2. Outline • Library preparation • Hybridization • cDNA expression arrays • Oligo expression arrays • Agilent • Affymetrix • Illumina • NimbleGen • Other array types

  3. Preparing a cDNA Library from mRNA

  4. Hybridization

  5. Glass Slide Microarrays (1994)

  6. Printing Glass Slide Arrays

  7. Synthetic Oligonucleotide Arrays Up to 25 bases

  8. Affymetrix Probes Schematic Actually Probes are (pseudo-) randomized

  9. Affymetrix Probe Sets • Probes for older expression arrays are drawn from the 3’ end of the gene • Poly-T priming picks up poly-A tails of transcripts • Newer exon and whole-gene arrays have probes evenly distributed • Random priming more even – but not uniform!

  10. Printed Oligonucleotide Arrays • Agilent (off-shoot of HP) uses printing technology

  11. Agilent Arrays • Now second largest supplier of arrays • Reputation for high quality and attention to detail (e.g. scanner optics) • Typical 60 nucleotide probes (60-mers) • 44K, 185K, and 244K standard sizes • Can do several (up to 8) arrays per slide

  12. NimbleGen Oligonucleotide Arrays Nimblegen uses a micro-mirror method to de-protect during oligo synthesis in situ

  13. (Roche-) NimbleGen Arrays • Usually 60-mers • Random sequence controls provided • Standard sizes from 385K up to 2.1 million probes • Can also be multiplexed • Patent issues kept the production facility in Iceland

  14. Illumina Bead Arrays • 3 mm beads manufactured with identifying segment (~12 nt) and 50-mer probe for target • Beads in wells (for some assays with optical fiber) • First scan reads ID tag; second reads target

  15. Illumina Probes • Typically about 30 beads per array • SD very high • No controls on most arrays • Can be multiplexed

  16. BioConductor • R packages for high-throughput biology • Robert Gentleman’s project 2000-2009 • Now run by steering committee • Mostly Object-Oriented (“S4”) • Easy to use interfaces • >source("http://bioconductor.org/biocLite.R")>biocLite() # will install most common packages • >biocLite(‘package name’) # for new ones

  17. Reading in Affymetrix Data • library(affy) • cel.data <- ReadAffy() • Look at the data: • The intensity() accessor gives the raw intensities for all values as laid out on chip • The pm() accessor gives the intensities for the ‘Perfect Match’ (i.e. functional) probes organized by probeset (alphabetically)

  18. Other Array Types

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