Gene Expression and the Transcriptome

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Gene Expression and the Transcriptome
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• The transcriptome is the complete set of
  transcripts and their relative levels of
  expression in a particular cell of tissue type
  under defined conditions.
• 1. DNA microarray
• 2. Serial analysis of gene expression (SAGE)
  

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22000 genes
Transcripts ??
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Parallel Analysis of Gene Expression Microarray
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4.1 (Part 1) Flow diagram for gene expression
profiling
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4.1 (Part 2) Flow diagram for gene expression
profiling
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Microarray Technologies

• cDNA microarray
• Oligonucleotide microarray
• 1. Long oligonucleotide
• 5070 mer
• 2. Short oligonucleotide
• 2030 mer

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Methods for RNA extract
Glass fiber-based extraction RNeasy, Qiagen 96
well RNeasy One-step disruption/extraction
solutions TRIZOL RNA Bee
www.qiagen.com
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4.7 (Part 1) Three methods for labeling cDNA
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cDNA microarray
cDNA
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Oligonuclotide microarray

• In oligonucleotide microarrays, short DNA
  oligonucleotides are spotted onto the array or
  synthesized in situ.
• The main feature of oligonucleotide microarray is
  that each gene is normally represented by more
  than one probe the ensemble of probes mapping to
  different regions of the gene is usually called
  probe set.

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Synthesis of Oligonucleotide
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4.5 Principle of oligonucleotide arrays
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4.6 (Part 1) Construction of oligonucleotide
arrays
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4.4 Types of printing pins
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Solid pin
Quill pin
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4.8 Microarray images produced with a
pin-and-loop arrayer
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Scatter plots
classical scatter plot
M-A plot for microarray analysis
M
A
Differentially expressed genes are higher (or
lower) in one of the samples Use an appropriate
cut-off (distance from diagonal) to select
relevant genes ? highly arbitrary!
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4.9 Simple normalization of microarray data
8 genes case
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4.8 Microarray images produced with a
pin-and-loop arrayer
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Exercise 4.2 (Part 1) Calculate which genes are
differentially expressed
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Exercise 4.2 (Part 2) Calculate which genes are
differentially expressed
Two-fold change log(2/1) or log(1/2)
Sample 1
Sample 2
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t-test statistical significance of observed
difference
T-test

• requires independent experimental replication
• assumes the data are identically normally
  distributed

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Exercise 4.3 Evaluate the significance of the
following gene expression differences
For Gene 1
(287198366255402)/5
(606441702597888)/5
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df55-28
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• 2. Hierarchical cluster analysis
• Euclidean distance
• Pearson correlation

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d1(34) 0.5(d13d14)
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d(15)(34) 0.25(d13d14 d35 d45)
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Exercise 4.4 Perform a cluster analysis on gene
expression profiles
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4.12 Hierarchical clustering of gene expression
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4.15 (Part 1) Principle of SAGE
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4.15 (Part 2) Principle of SAGE
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4.18 (Part 1) Quantitative RT-PCR
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4.18 (Part 2) Quantitative RT-PCR
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4.14 (Part 1) Chromatin immunoprecipitation and
regulatory pathways