mb206 1. also known as dna chip allows simultaneous measurement of the level of transcription for...
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MB206 1
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Also known as DNA ChipAllows simultaneous measurement
of the level of transcription for every gene in a genome (gene expression)
Transcription? Process of copying of DNA into
messenger RNA (mRNA) Environment dependant!
Microarray detects mRNA, or rather the more stable cDNA
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• High-throughput measuring- 5000-20000 gene expressions at the same time
• Identify genes that behaves different in different cell populations- tumor cells vs healthy cells- brain cells vs liver cells- same tissue different organisms
• Time series experiments- gene expressions over time after treatment
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microarray
scanning
analysis
cDNA clones(probes)
PCR product amplificationpurification
printing
0.1nl / spotHybridize
RNA
Tumor sample
cDNA
RNA
Reference sample
cDNA
excitation
red lasergreen
laser
emission
overlay images and normalise
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Hybridize
RNA
Tumor sample
cDNA
RNA
Reference sample
cDNA
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Biological questionDifferentially expressed genesSample class prediction etc.
Testing
Biological verification and interpretation
Microarray experiment
Estimation
Experimental design
Image analysis
Normalization
Clustering Discrimination
R, G
16-bit TIFF files
(Rfg, Rbg), (Gfg, Gbg)
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Laser scans array and produces images One laser for each color, e.g. one for green,
one for red Image analysis, main tasks:
Noise suppression Spot localization and detection, including the
extraction of the background intensity, the spot position, and the spot boundary and size
Data quantification and quality assessment Image Analysis is a book on its own:
Kamberova, G. & Shah, S. “DNA Array Image Analysis Nuts & Bolts“. DNA Press LLC, 2002
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Transformed data {(M,A)}n=1..5184:
M = log2(R/G) (ratio),
A = log2(R·G)1/2 = 1/2·log2(R·G) (intensity signal)
R=(22A+M)1/2, G=(22A-M)1/2
“Observed” data {(R,G)}n=1..5184:
R = red channel signalG = green channel signal
(background corrected or not)
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Biased towards the green channel & Intensity dependent artifacts
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Scaled print-tip normalization
Median Absolute Deviation (MAD) Scaling
Averaging
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Extreme in T values?
Extreme in M values?...or extreme in some other statistics?
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Gene: Mavg Aavg TSE
2341 -0.86 10.9 -18.00.125
6412 -0.75 11.1 -14.70.102 6123 -0.70 9.8-12.2 0.121
102 0.65 10.3 -14.50.136 2020 0.64 9.3 -11.90.118
3132 0.62 9.9 -14.40.090
4439 -0.62 9.7 -14.60.088
2031 -0.61 10.7 -13.70.087
657 -0.60 9.2 -13.60.094
502 0.58 10.0 -12.70.101
1239 -0.58 9.8 -11.40.103
5392 -0.57 9.9 -20.70.057
3921 0.52 11.3 13.50.083
...
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10. Which genes are actually up- and down regulated?
11. P-values.
12. Planning of experiments:- what is best design?- what is an optimal sample sizes?
13. Classification:- of samples.- of genes.
14. Clustering:- of samples.- of genes.
15. Time course experiments.
16. Gene networks.- identification of pathways
17. ...
1. Image analysis- what is foreground?- what is background?
2. Quality- which spots can we trust?- which slides can we trust?
3. Artifacts from preparing the RNA, the printing, the scanning etc.
4. Data cleanup
5. Normalization within an experiment:- when few genes change.- when many genes change.- dye-swap to minimize dye effects.
6. Normalization between experiments:- location and scale effects.
7. What is noise and what is variability?
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MB206 20
Brown & Botstein, 1999