Serial Analysis of Gene Expression

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Serial Analysis of Gene Expression

• Velculescu, V., Zhang, L., Vogelstein, B.
  Kinzler, K. (1995) Science

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Authors The Wild-Types

• Victor VelculescuKeyboard
• Ken KinzlerDrummer
• Bert VogelsteinKeyboard
• Lin ZhangHonorary member

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Current Research
Victor Velculescu Dept. of Oncology, Johns
Hopkins University. -New therapeutic targets for
colon cancer Lin Zhang Dept. of Pharmacology,
University of Pittsburg. -genetic basis of
differential sensitivity to anticancer drugs, as
well as the genetic alterations in cancer cells
that cause drug resistance Bert Vogelstein Dept.
of Oncology, Johns Hopkins University. -Identifica
tion and characterization of oncogenes. Ken
Kinzler Dept. of Oncology, Johns Hopkins
University.
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Aim of This Paper

• Provide researchers with a means to study
  expression of an entire genome rather than single
  genes
• To create a method of comparing gene expression
  differences between two samples with information
  about abundance of transcripts
• To illustrate effectiveness of this technique by
  examining pancreatic gene expression patterns
• To confirm the quantitative nature of the novel
  technique through library screening

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Major Findings

• Developed a new technique to study gene
  expression that is capable of quantitative
  analysis of an entire genome
• Characterized gene expression in human pancreas
• Used SAGE to identify novel cDNA transcripts

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SAGE Based on 2 principles

1. A 9 bp tag is sufficient to unambiguously
   identify a gene
2. Concatenation (linking together) of these short
   DNA sequences increases the efficiency of
   identifying unique transcripts in a serial
   manner.

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Methods

1. mRNA cDNA
2. Cleave with A.E
3. Isolate 3 most transcript of each cDNA by
   binding to Streptavidin beads
4. Divide cDNA in half
5. Ligate to 1 of 2 linkers (each with a T.E site)
6. Ligate the two pools of tags together.

FIG. 1 SAGE protocol
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Methods

1. Ligated linkers serve as primers for
   amplification
2. Cleave PCR products with A.E. to isolate ditags
3. Concatenate by ligation
4. Clone
5. Sequence

FIG. 1 SAGE protocol
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SAGE Analysis Human Pancreas
Table 1. Pancreatic SAGE tags.
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Comparison of Transcript Abundance

• Quantitative nature of SAGE tested by
  construction of a pancreatic cDNA library that
  was screened with cDNA probes for several
  pancreatic genes
• Relative abundance of SAGE tags agreed with
  results from library screening

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Current Applications

• Gene Discovery
• Analysis of Cardiovascular gene expression
• Gene expression in carcinogenesis
• Substance abuse studies
• Cell, tissue and developmental stage profiles in
  C. elegans
• Profiling of human diseases
• and more..

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Other Technologies to Study Expression

1. Expressed Sequence Tags (ESTs)
2. RT-PCR
3. RNA Blotting
4. DNA Microarrays

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Expressed Sequence Tags

• Short, annotated sequences at 3 or 5 end
• Can be used to determine the number of
  genes/genome
• Can be used to identify novel genes

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qRT-PCR

• Can be used to both detect quantify gene
  expression
• Not high-throughput
• Can only be used on a limited scale

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RNA Blotting

• To quantify RNA (semi-quantitative)
• Can detect RNA slicing
• Again, not quantitative

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DNA microarrays

• High-throughput
• Allows for simultaneous detection of genome-wide
  expression
• Can provide relative quantitative information
  about expression

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Microarray vs. SAGE
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C. elegans temporal Tissue-Specific Expression
Profiling Project

• About 1.9 million C.elegans tags sequenced to
  date
• Several libraries have been completed, including
  Embryo, all larval stages, Young and old adult,
  sterile adult, microdissected gut tissue,
  FACS-sorted muscle cells, and FACS embryonic
  intestinal cells
• Several libraries are in progress, including
  FACS-sorted pan-neural cells, FACS-sorted
  ciliated neurons, FACS pharynx, and FACS
  hypodermal cells

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Long SAGE vs. Short SAGE

• A comparison of short SAGE (14bp) vs. long SAGE
  (21bp)
• Some tags are not unambiguously assigned to a
  gene (similar 3 ends due to ancestral
  duplications)
• About 12 of C. elegans tags are not
  unambiguously identified using 14bp tags
• Results of empirical data suggests that LongSAGE
  gives far greater resolution, but at an increased
  cost.

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Why is this paper a landmark?

1. New technique for quantitative analysis of gene
   expression
2. Identified novel transcripts in pancreatic
   samples
3. Has allowed for the characterization of gene
   expression, both temporally and spatially, in
   several organisms
4. Currently is being used to study disease (i.e.
   cancer) gene expression

sciencepark.mdanderson.org/ ggeg/SAGE_technique.h
tm
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References

• Velculescu, V., Zhang, L., Vogelstein, B.
  Kinzler, K. (1995). Serial Analysis of Gene
  Expression Science 270 (5235)
• Reinke, V. (2002). Functional Expolation of the
  C. elegans Genome Using DNA Microarrays. Nature
  Genetics 32

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