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

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

8. “Long Oligos”
Like cDNAs, but instead of using a cloned gene, design a 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
24~50µm
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 Set
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 5’ 3’ Probe Set Perfect Match Mismatch
Twenty oligo probes are selected from the last 600 bases from the 3’ end of the gene
Perfect Match
Mismatch
25 mer DNA oligo
For each probe selected, a partner containing a central mutation is also made
Perfect Match
Mismatch
Probe Set
Probe Pair PM MM
Probe Cell
24m
For each gene a total of 20 probe pairs are arrayed on the chip

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

16. IVT cRNA synthesis amplifies and labels transcripts with Biotin
cRNA preparation
Total RNA (5-8 mg)
AAAAAAAAA
cDNA Strand 1 synthesis
TTTTTTTTTNNNNNNNNN
AAAAAAAAA
SS II reverse transcriptase
T7RNA pol. promoter
cDNA Strand 2 synthesis
TTTTTTTTTNNNNNNNNN
AAAAAAAAA
NNNNN
E. coli DNA pol. I
T7RNA pol. promoter
IVT cRNA synthesis amplifies and labels transcripts with Biotin
NNNNNNNNNNNNN
AAAAAAAAAAAAAAN
TTTTTT T
UUUUUUUUUU
……….. ……
…….
T7 RNA pol.
NNNNNNNN
SS II reverse transcriptase may not finish the job that’s why the 3’ end of the DNA are chosen as the probes
In Vitro Transcription (IVT) Synthesis
Fragmented cRNA
cRNA is now ready for hybridization to test chip

17. Post hybridiz-ation washes
cRNA labeled targets
Post hybridiz-ation washes
Non-Specific Binding
Specific Binding B
cRNA labeled targets B S FL
cDNA probes
B—biotin
S—streptavidin
Biotinylated cRNA was then generated from the cDNA by an in vitro transcription reaction in which biotin-11-CTP and biotin-16-UTP were included.
FL-- fluorescent

18. S FL B B S FL B S FL S FL
Streptavidin

19. Microarray experiment
cDNA
IVT
(B-UTP) B
Biotin-Labeled
cRNA transcript
Cells
Poly (A)+
RNA
AAAA
Fragment
(heat, Mg2+)
Biotin-Labeled
cRNA fragments B
Hybridize
(1-18 hours)
Wash
Stain
Scan

20. The chip image data file (or “
The chip image data file (or “.dat” file) is the first part of data acquisition and appears on the computer screen upon completion of the laser scan.
.dat file
Here, we zoom in to see an individual probe set that has been highlighted
Probe set

21. The first image is “sample1. dat
The first image is “sample1.dat.” note the pixel to pixel variation within a probe cell
A “*.cel.” file is automatically generated when the “*.dat” image first appears on the screen. Note that this derivative file has homogenous signal intensity within its probe cells
.cel file

22. Affymetrix Algorithms 1. Signal
1.1 Adjusting MMs to purge negative values
All MMs < PMs, No adjustment necessary
Few MMs > PMs, change MMs based on weighted mean of other MMs
Most MMs > PMs, change MMs to be slightly lesss than PM

23. Affymetrix Algorithms
Signal Calculation. Calculate the signal PM MM
Having adjusted the MM values, we now calculate the signal
PM-MM
Unweighted mean = 2063
The PM values.
Standard deviations 1
Weight factor
The unweighted mean is vulnerable to outlier data. In order to protect against this, we dampen the effect of outliers by using the Tukey bi-weight mean. PM-MM values that are a number of standard deviations away from the mean are given low weights in accordance with the graph shown here. Individual PM-MM data are multiplied by the weight factor before calculation of the mean. The weighted mean is then called the “signal.”
The MM values.
The PM-MM values are calculated.
Using Tukey’s 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 27 11 38 2 1 27 11