English okay

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English okay?   Masters studies offer
tracks This is part of VL Microarray data
analyis Tuesday, 830 1000 Ü Thursday
1015-1145 (start Oct. 23) Next semester
Praktikum Seminar Thereafter possibility for
Masters thesis.    Anwesenheitspflicht in VL und
Ü (Liste!)   Literature See course web page.  
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• 21. Okt Microarray-Technologien Martin Vingron
• 28. Okt Grundlagen der Datenanalyse Christine
  Steinhoff
• 4. Nov Varianzanalyse I Christine Steinhoff
• 11. Nov Varianzanalyse II Christine Steinhoff
• 18. Nov LOWESS, Varianzstabilisierung Anja von
  Heydebreck
• 25. Nov Statistisches Testen Anja von
  Heydebreck
• 2. Dez Clusterverfahren Anja von Heydebreck
• 9. Dez Klassifikation, Lin. Diskriminanzanalyse Ra
  iner Spang
• 16. Dez Anwendungen in der Krebsforschung Rainer
  Spang
• 6. Jan Hauptkomponentenanalyse Martin Vingron

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Determination of DNA sequence Derivation of amino
acid sequences Analysis, comparison,
classification
Study of gene function gene expression studies
proteomics metabolic networks
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DNA
gene
transcription
messenger RNA (mRNA)
translation
protein
sequence
structure
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A cell and its population of genes
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What is the problem?

• Determine the amount of mRNA for each
• gene that is present in a cell/tissue.

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DNA forms double strands by a process
called hybridization
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Labeling
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Hybridization
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Expression Arrays
cDNA Arrays
Oligonucleotide Arrays
Glas Arrays
Membrane based Arrays
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Glass Slide Microarrays
were first produced at Stanford University
(Schena et al, 1995).
Whole cDNA 500-1500 bp
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Filter Macroarrays
were first published by Lennon and Lehrach, 1991
7.5x2.5cm
Ca 21 cm
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Oligonucleotide Arrays
were first published by Lockhardt et al, 1996
... TGTGATGGTGGGAATGGGTCAGAAGGACTCCTATGTGGGTGACGAG
GCC
TTACCCAGTCTTCCTGAGGATACAC
TTACCCAGTCTTGCTGAGGATACAC
ca 25bp
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Probe - Reference
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There are other technologies, too, to estimate
expression levels

• EST sequencing electronic northern
• SAGE tags of mRNAs are concatenated and
  sequenced
• Reliability of results depends on depth of
  probing (number of ESTs, number of tags)

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Why do we want to know?

• tissue profiling which genes are expressed in
  a tissue
• Comparing healthy and diseased (e.g., tumor)
  tissue
• Studying dynamic processes E.g., cell cycle
  (time series)

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Example Renal clear cell carcinoma

• Comparison of kidney cancer cells to normal
  tissue. Which genes are altered in their
  expression?

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N98-8880
T98-8880
Molecular Genome
Analysis Dr.
Judith Boer
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Example Cell cycle time course
G1 S G2
M
Spellman et al took several samples per
time-point and hybridized the RNA to a glass
chips with all yeast genes
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Data processing

• Image collection
• Image analysis, intensity determination
• Within slide normalization

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OUPUT Scanner Scanner-Software
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Different technologies

• Support membrane or glass slide
• Spotted material PCR product or oligo
  (short/long)
• Labeling
• 1-channel radioactive, Affy
• Absolute values
• 2-channel 2 color fluorescent labeling
• Relative values

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Quality issues
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subpopulations PCR
Remedies improve PCR protocols model random
effect through plate-wise calibration
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subpopulations pin
Remedies handling of pins pin-wise calibration
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Distribution of intensities log-normal?
intensities
log intensities
QQPlot
Histogramm
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Chip design

• Type of chip
• Global whole genome (yeast, drosophila, mouse,
  man)
• Domain specific, e.g. cancer, infection
• Spots
• PCR products E.g., 3 UTR (avoid crosshyb.)
• Oligos uniqueness, stability

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Databases

• Stanford
• TIGR
• Gene expression atlas
• GEO
• Arrayexpress
• MIAME standard Minimum Information About a
  Microarray Experiment

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Software

• R Bioconductor
• Jexpress
• Genesprings
• Rosetta Resolver

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Affymetrix technology

• Per gene, spot 20 perfectly matching oligos and
  20 oligos with 1 mismatch
• Intensity weighted average of pixel intensities
  in perfect and mismatch oligos
• (More on this next week)