Genomics techniques: An overview of methods for the study of gene expression

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• Genomics techniques An overview of methodsfor
  the study of gene expression
• D. E. Moody 2001 J. Anim. Sci. 79(E.
  Suppl.)E128E135

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The objectives

• The tools of massive screening of global gene
  expression
• The principal of each technique
• The advantage and limitation of each technique
• The application of each technique

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Evaluate global gene expression

• Differential displaying
• Suppressive subtractive hybridization (SSH)
• Sequencing of expressed sequence tags (EST),
  serial analysis gene expression (SAGE Long
  SAGE)
• Hybridization to microarrays

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Subtractive hybridization

• Gurskaya et al. (1996) and Diatchenko et al.
  (1996) described the protocols, termed
  sup-pression subtractive hybridization (SSH).
• The SSH method is designed to selectively amplify
  differentially expressed transcripts while
  suppressing the amplification of abundant
  transcripts, thus eliminating the need to
  separate single- and double-stranded molecules.

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Figure 4. Typical result of positive control
skeletal muscle ds cDNA synthesis. before (Lane
1) and after (Lane 2) Rsa I digestion. cDNA was
synthesized as described in the protocol from the
human skeletal muscle control poly A RNA. Lane
M DNA size markers.
_at_ After Rsa I digestion, the average cDNA size is
smaller (0.12 kb compared to 0.510 kb).
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Figure 5. Typical results of the ligation
efficiency analysis. Lane 1 PCR products using
Tester 1-1 (Adaptor 1-ligated) as the template
and the G3PDH 3' Primer and PCR Primer 1. Lane 2
PCR products using Tester 1-1 (Adaptor 1-ligated)
as the template, and the G3PDH 3' and 5' Primers.
Lane 3 PCR products using Tester 1-2 (Adaptor
2R-ligated) as the template, and the G3PDH 3'
Primer and PCR Primer 1. Lane 4 PCR products
using Tester 1-2 (Adaptor 2R-ligated) as the
template, and the G3PDH 3' and 5' Primers. 2
agarose/ EtBr gel. Lane M fX174 DNA/ Hae III
digest size markers.
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Figure 6. Typical results of the control skeletal
muscle subtraction experiment. Lane 1
Secondary PCR products of subtracted skeletal
muscle tester cDNA containing 0.2 fX174/ Hae III
digested DNA. Lane 2 Secondary PCR products of
unsubtracted skeletal muscle tester cDNA ligated
with both Adaptors 1 and 2R (generated in Section
IV.F) and containing 0.2 fX174/ Hae III-digested
DNA. 2 agarose/EtBr gel.
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Limitations

• SSH remains applicable only to pair-wise
  treatment comparisons and must be replicated with
  the tester and driver reversed to identify gene
  expression changes in both directions.
• Additionally, subtractive hybridization does not
  provide a quantitative measure of expression
  differences and is most efficient at identifying
  genes that are completely absent, rather than
  expressed less abundantly, in the driver sample.

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Advantages
Differential display

• to compare multiple experimental samples
  simultaneously
• to identify genes that are either up or
  down-regulated in one sample relative to another.

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Differential display

• Two significant limitations of the original
  protocol were
• the requirement of large quantities of mRNA
  (e.g., 270 µg by Sargent and Dawid, 1983)
• a bias against the identification of rare
  transcripts.
• not a quantitative method with a high rate of
  false positives, or
• gene fragments that seem to be differentially
  expressed as an artifact

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Expressed sequence Tag Sequencing

• The EST sequences are generated by randomly
  picking clones from a cDNA library and performing
  a single sequencing reaction to produce 300 to
  500 bp of sequence per clone.
• Differences in gene expression may be identified
  by counting the number of times a particular
  sequence appears in EST libraries of genes from
  different sources.

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Homo sapiens
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NCKU Xcrunch to perform Digital Differential
Display and Digital Northern
Bladdercancer
Normal Bladder
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Serial Analysis of Gene Expression

• Serial analysis of gene expression (SAGE
  Velculescu et al., 1995) is a technique for the
  analysis of gene expression that is essentially
  an accelerated version of EST sequencing.
• Typically, a SAGE tag includes nine bases of
  sequence downstream from the last endonuclease
  recognition site of a transcript. Multiple SAGE
  tags are ligated together in a cloning vector so
  that a typical sequencing reaction of 300 to 500
  bp generates the sequences of 20 to 30 SAGE tags

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Serial Analysis of Gene Expression

• Advantages
• SAGE data are quantitative and cumulative.
• Accurate, quantitative transcript profiles
  describing the abundance of all genes expressed
  in a cell or tissue are generated by SAGE, if
  sufficient sequencing is completed.

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Long SAGE

• 17-21 bp tags created by type IIS restriction
  endonuclease Mme I to create a 17 bp fragment.
  99.8 tags occur only once in the whole genome.
• Super SAGE gt 25 bp tag, created by type III
  restriction enzyme EcoP15I.
• SAGE 10-14 bp tags created by type IIS
  restriction endonuclease Nla III.

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Human RBBP7

• Size 424 amino acids, M.W 47820
• Function Core-histone-binding subunit that
  target chromatin assembly factor, chromatin
  remodeling factor, HATs and HDACs to their
  histone substrates in a manner that is regulated
  by nucleosomal DNA.
• Subcellular location Nuclear.
• Similarity Contains 6 WD Repeats (TRP-ASP
  domains)

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Similar Genes in other Organisms
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RBBP7 expression in normal human tissues (based
on quantifying ESTs from various tissues in
Unigene clusters)
Prostate, Kidney, Lung, Bone marrow
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SAGE Anatomic Viewer Results
High expression (in normal)
Prostate , Kidney Cancer expression
Increased Brain , Stomach , Pancreas , Colon
,Peritoneum Decreased Prostate
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SAGE Brain Anatomic Viewer Results
RBBP7 expression increased in brain cancer tissues
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NCI60 NCI 60 Microarray Expression Data
RBBP7
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Digital Northern Results
293T
MCF7
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Virtual Northern
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Microarray Hybridization

• The analysis of array data may be divided into
  three components
• 1) identification and quantification of
  hybridization intensities,
• 2) visualization of data, and
• 3) clustering techniques
• Based on the assumption that genes with related
  functions are coregulated, clustering of
  microarray data becomes a powerful method to
  assign putative functional classifications to
  novel genes.

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• No single technique is optimal for all
  applications, and the optimal strategy for
  specific situations may prove to be creative
  combinations of several techniques.

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Bladder Cancer Cell linesE6?RT4?TSGH8301?J82
High Throughput Real-Time PCR
Putative markers for Bladder Cancer
Animal Models
Verification by clinical samples
Cell line system
Establish a Website of Bladder Cancer,http//apple
.csie.ncku.edu.tw/bladder/
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Microarray raw data
Platform for Cancer-related microarray service
Data conversion and integration
Data Analysis
Ontology analysis
Cancer-related Primer seq. database
Clone purchasing
NCKU clone library
Cancer-related gene primer set
Cell-line cDNA bank
Cancer specific customized cDNA chip or
oligonucleotid chip
Tumor tissue cDNA bank