Transcript Serial Analysis of Gene Expression

Serial Analysis of Gene
Expression
Velculescu, V., Zhang, L., Vogelstein,
B. Kinzler, K. (1995) Science
Authors: The Wild-Types
•Victor Velculescu:
Keyboard
•Ken Kinzler
Drummer
•Bert Vogelstein
Keyboard
•Lin Zhang
Honorary member
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.
-Identification and characterization of oncogenes.
Ken Kinzler: Dept. of Oncology, Johns Hopkins University.
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
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
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.
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
Methods
7. Ligated linkers serve as
primers for amplification
8. Cleave PCR products
with A.E. to isolate
ditags
9. Concatenate by ligation
10. Clone
11. Sequence
FIG. 1 SAGE protocol
SAGE Analysis: Human Pancreas
Table 1. Pancreatic SAGE tags.
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
Current Applications
1. Gene Discovery
2. Analysis of Cardiovascular gene expression
3. Gene expression in carcinogenesis
4. Substance abuse studies
5. Cell, tissue and developmental stage profiles in C.
elegans
6. Profiling of human diseases
and more…..
Other Technologies to Study Expression
1. Expressed Sequence Tags (ESTs)
2. RT-PCR
3. RNA Blotting
4. DNA Microarray’s
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
qRT-PCR
• Can be used to both detect
& quantify gene expression
• Not high-throughput
• Can only be used on a
limited scale
RNA Blotting
•To quantify RNA (semi-quantitative)
• Can detect RNA slicing
• Again, not quantitative
DNA microarrays
• High-throughput
• Allows for simultaneous
detection of genome-wide
expression
• Can provide relative
quantitative information about
expression…
Microarray vs. SAGE
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
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.
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.htm
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