Serial analysis of gene expression

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Summary of SAGE

Serial Analysis of Gene Expression (SAGE) is a technique used in molecular biology to measure the expression levels of genes. It was developed in the mid-1990s and allows for the comprehensive analysis of gene expression patterns in a given cell or tissue type.

Principle

The principle behind SAGE is to generate short sequence tags (10-14 base pairs) that correspond to fragments of the mRNA transcripts present in the sample. These tags are then concatenated into long DNA molecules, which can be sequenced to identify the abundance of each transcript. The short tags are sufficient to uniquely identify the corresponding gene, allowing for the quantification of gene expression levels.

Methodology

The SAGE process involves several key steps:

1. Isolation of mRNA: The first step is to isolate mRNA from the cells or tissues of interest.
2. cDNA Synthesis: The mRNA is then reverse transcribed into complementary DNA (cDNA).
3. Tag Generation: Short sequence tags are generated from the cDNA using restriction enzymes.
4. Tag Concatenation: The tags are ligated together to form long concatemer molecules.
5. Sequencing: The concatemers are sequenced using high-throughput sequencing methods.
6. Data Analysis: The sequence data is analyzed to count the frequency of each tag, which corresponds to the expression level of the associated gene.

Applications

SAGE has been widely used in various fields of biological research, including:

• Cancer research: To identify genes that are differentially expressed in cancerous tissues compared to normal tissues.
• Developmental biology: To study gene expression patterns during different stages of development.
• Neuroscience: To analyze gene expression in different regions of the brain.

Advantages and Limitations

Advantages

• High-throughput: SAGE allows for the simultaneous analysis of thousands of genes.
• Quantitative: Provides a quantitative measure of gene expression levels.
• Unbiased: Does not require prior knowledge of the genes being studied.

Limitations

• Complexity: The technique involves multiple steps and can be technically challenging.
• Cost: High-throughput sequencing can be expensive.
• Short Tags: The short length of the tags can sometimes lead to ambiguity in gene identification.

Related Techniques

• RNA-Seq
• Microarray
• Northern blot

See Also

• Gene expression
• Transcriptomics
• Functional genomics