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DNA microarray and array data analysis

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DNA microarray is a new technology to measure the level of the mRNA gene ... In order to protect against this, we dampen the effect of outliers by using the ... – PowerPoint PPT presentation

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Title: DNA microarray and array data analysis


1
DNA microarray and array data analysis
  • Some of the slides are adapted from the lecture
    notes of Dr. Patrick Leahy of the Gene Expression
    Array Core Facility at CWRU

2
(No Transcript)
3
What is DNA Microarray
  • DNA microarray is a new technology to measure the
    level of the mRNA gene products of a living cell.
  • A microarray chip is a rectangular chip on which
    is imposed a grid of DNA spots. These spots form
    a two dimensional array.
  • Each spot in the array contains millions of
    copies of some DNA strand, bonded to the chip.
  • Chips are made tiny so that a small amount of RNA
    is needed from experimental cells.

4
DNA Microarray
  • Many applications in both basic and clinical
    research
  • determining the role a gene plays in a pathway,
    disease, diagnostics and pharmacology,
  • There are three main platforms for performing
    microarray analyses.
  • cDNA arrays (generic, multiple manufacturers)
  • Oligonucleotide arrays (genechips) (Affymetrix)
  • cDNA membranes (radioactive detection)

5
cDNA Microarray
  • Spot cloned cDNAs onto a glass/nylon microscope
    slide
  • usually PCR amplified segments of plasmids
  • Complementary hybridization
  • -- CTAGCAGG actual gene
  • -- GATCGTCC cDNA (Reverse transcriptase)
  • -- CUAGCAGG mRNA
  • Label 2 mRNA samples with 2 different colors of
    fluorescent dye -- control vs. experimental
  • Mix two labeled mRNAs and hybridize to the chip
  • Make two scans - one for each color
  • Combine the images to calculate ratios of amounts
    of each mRNA that bind to each spot

6
Spotted Microarray Process
CTRL
TEST
7
cDNA Array Experiment Movie
  • http//www.bio.davidson.edu/courses/genomics/chip/
    chip.html

8
Long Oligos
  • Like cDNAs, but instead of using a cloned gene,
    design a 40-70 base probe to represent each gene
  • Relies on genome sequence database and
    bioinformatics
  • Reduces cross hybridization
  • Cheaper and possibly more sensitive than Affy.
    system

9
Affymetrix
  • Uses 25 base oligos synthesized in place on a
    chip (20 pairs of oligos for each gene)
  • cRNA labeled and scanned in a single color
  • one sample per chip
  • Can have as many as 47,000 probes on a chip
    (HG-U133 Plus 2.0 Array)
  • Arrays get smaller every year (more genes)
  • Chips are expensive (about 400/chip)
  • Proprietary system black box software, can
    only use their chips

10
Affymetrix Genome Arrays
11
Affymetrix GeneChip Probe Array
12
Affymetrix GeneChip Probe Arrays
Hybridized Probe Cell
GeneChip Probe Array
Single stranded, fluorescently labeled cRNA target
Oligonucleotide probe
2450µm
1.28cm
Each probe cell or feature contains millions of
copies of a specific oligonucleotide probe
Image of Hybridized Probe Array
BGT108_DukeUniv
13
Affymetrix GeneChip
  • Probe
  • 25 bases long single stranded DNA oligos
  • Probe Cell
  • Single square-shaped feature on an array
    containing one type of probe.
  • Contains millions of probe molecules
  • Probe Pair
  • Perfect Match/Mismatch

14
Array Design
15
Probe Sub-types on chips
  • Known genes
  • Specific transcripts
  • Exemplars
  • Consensus
  • Housekeeping genes
  • Expressed sequence tags (ESTs)
  • Spiked control transcripts

16
cRNA preparation
NNNNNNNN
cRNA is now ready for hybridization to test chip

17
(No Transcript)
18
Streptavidin
19
Microarray experiment
20
.dat file
21
.cel file
22
Affymetrix Algorithms
1. Signal
1.1 Adjusting MMs to purge negative values
All MMs lt PMs, No adjustment necessary
Few MMs gt PMs, change MMs based on weighted mean
of other MMs
Most MMs gt PMs, change MMs to be slightly lesss
than PM
23
Affymetrix Algorithms
Signal Calculation. Calculate the signal
PM 1000 5000 430 765 355
98 3005 413 20333 590
MM 900 2000 230 25 331
40 1200 203 6197 230
PM-MM 100 3000 200 740
24 58 1805 210 14136 360
Unweighted mean 2063
Using Tukeys biweight mean 1780 Signal
(expression level) 1780
24
.xls file
25
ALL_vs_AML_train_set_38_sorted.res
26
ALL_vs_AML_train_set_38_sorted.cls
  • 38 2 1
  • 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
    0 0 1 1 1 1 1 1 1 1 1 1 1

27
11
27
(No Transcript)
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