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Bioinformatics for Big Omics Data: Introduction to gene expression microarrays

Raphael Gottardo
January 21, 2014

Setting up some options

Let's first turn on the cache for increased performance and improved styling

# Set some global knitr options
library("knitr")
opts_chunk$set(tidy=TRUE, tidy.opts=list(blank=FALSE, width.cutoff=60), cache=TRUE, messages=FALSE)

Outline

  • Motivation
  • Reverse transcription and hybridization cDNA microarrays
  • Oligonucleotide arrays (Affymetrix)
  • Illumina beadarrays

Motivation

  • Why are microarrays so important?
  • First technology to enable the genomewide quantification of gene-expression
  • Characterize and classify diseases, design new drugs, etc.
  • "Recent" technology (1995,1996)
  • Fast moving field

Microarrays

  • Isolate mRNA from cells
  • From RNA we can get DNA using an enzyme called reverse transcriptase (e.g. Retrovirus)
  • The derived "copy" DNA (cDNA) is hybridized to known DNA targets (genes) to quantify gene expression

Reverse transcription

Hybridization

cDNA-microarray

cDNA-microarray

Oligonucleotide arrays

  • Fabricated by placing short cDNA sequences (oligonucleotides) on a small silicon chip by a photolithographic process
  • Each gene is represented by a set of distinct probes, cDNA segments of length 25 nucleotides (11-20)
  • Probes chosen based on uniqueness criteria and empirical rules
  • Single color

Affymetrix arrays

Affymetrix arrays

  • about 20 probes that “perfectly” represent the gene (Perfect Match)

  • about 20 probes that do not match the gene sequence (Mismatch)

  • Probeset

Affymetrix arrays

Affymetrix arrays

For a valid gene expression measurement the Perfect Match sticks and the Mistach does not!

Probe synthesis

Let's watch a small video

http://www.youtube.com/watch?v=ui4BOtwJEXs

Affymetrix arrays

Affymetrix protocol

Affymetrix probesets

Affymetrix image

Illumina bead arrays

(Source: http://www.illumina.com/)

Illumina bead arrays

(Source: http://www.mouseclinic.de/)

Illumina bead arrays - image

Illumina bead arrays

  • Illumina bead arrays can be applied to different problems
  • Can use one or two colors depending on the application
  • Gene expression arrays use one color

Illumina bead arrays

  • High density arrays
    • HumanHT-12 v4.0 Expression BeadChip, about 47,000 probes
  • A specific oligonucleotide sequence is assigned to each bead type, which is replicated about 30 times on an array
  • Bead level data can be relavitely large

Summary of technologies

cDNA microarray Affymetrix Illumina
1 probe/gene 1-20 probes/gene 30 beads(probes)/gene
Probe of variable lengths 25-mer probes 50-mer probes
Two colors One color One color
Flexible, choice of probes High density/Replication High density/Replication
  • | Cross hybridization | -

Analysis pipeline

  1. Biological question
  2. Experimental design
  3. Experiment
  4. Image analysis
  5. Normalization, Batch effect removal
  6. Estimation, Testing, Clustering, Prediction, Classification, Feature selection, etc.
  7. Validate finding generate new hypothesis -> New experiments

Most of these steps require the use of statistical methods and/or computational tools

Note: Even though microarrays were designed to study gene expression, they can be used to study many other things (DNA-protein binding, methylation, splicing, etc). Though they are slowly being replaced by sequencing-based assays.