Serial Analysis of Gene Serial Analysis of Gene ExpressionExpression

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

Authors: The Wild-Types•Victor Velculescu:Keyboard

•Ken KinzlerDrummer

•Bert VogelsteinKeyboard

•Lin ZhangHonorary member

Current ResearchVictor 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 principlesSAGE: Based on 2 principles1. 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.

Methods1. 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

Methods7. 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 Applications1. 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 Expression1. Expressed Sequence Tags (ESTs)

2. RT-PCR

3. RNA Blotting

4. DNA Microarray’s

Expressed Sequence TagsExpressed 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-PCRqRT-PCR• Can be used to both detect & quantify gene expression

• Not high-throughput

• Can only be used on a limited scale

RNA BlottingRNA Blotting•To quantify RNA (semi-quantitative)

• Can detect RNA slicing

• Again, not quantitative

DNA microarraysDNA 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 landmarkWhy 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