Serial analysis of gene expression (SAGE) is a technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest. The original technique was developed by Dr. Victor Velculescu at the Oncology Center of Johns Hopkins University, and was in the journal Science in 1995. Several variants have been developed since, most notably a more robust version, LongSAGE (developed by Dr. Saurabh Saha and colleagues at Johns Hopkins University), that enables annotation of existing genes and discovery of new genes within genomes. Jump to: navigation, search The life cycle of an mRNA in a eukaryotic cell. ... Jump to: navigation, search The Johns Hopkins University is a private institution of higher learning located in Baltimore, Maryland. ... Science is the journal of the American Association for the Advancement of Science (AAAS). ... Jump to: navigation, search 1995 was a common year starting on Sunday of the Gregorian calendar. ...

Overview

Briefly, SAGE experiments proceed as follows:

1. Isolate the mRNA of an input sample (e.g. a tumour).
2. Extract a small chunk of sequence from a defined position of each mRNA molecule.
3. Link these small pieces of sequence together to form a long chain (or concatamer).
4. Clone these chains into a vector which can be taken up by bacteria.
5. Sequence these chains using modern high-throughput DNA sequencers.
6. Process this data with a computer to count the small sequence tags.

A more in-depth, technical explanation of the technique is available here. The interaction of mRNA in a eukaryote cell. ... Tumor (American English) or tumour (British English) originally means swelling, and is sometimes still used with that meaning. ... The interaction of mRNA in a eukaryote cell. ... In the fields of molecular biology and genomics, a DNA sequencer is a machine for the automated determination of DNA sequences. ...

Analysis

The output of SAGE is a list of short sequence tags and the number of times it is observed. Using sequence databases a researcher can usually determine, with some confidence, the original mRNA (and therefore which gene) the tag was extracted from. In the field of bioinformatics, a sequence database is a large collection of DNA, protein, or other sequences stored on a computer. ... The interaction of mRNA in a eukaryote cell. ... Jump to: navigation, search This stylistic schematic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome (right). ...

Statistical methods can be applied to tag and count lists from different samples in order to determine which genes are more highly expressed. For example, a normal tissue sample can be compared against a corresponding tumour to determine which genes tend to be more (or less) active. Jump to: navigation, search This stylistic schematic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome (right). ... Biological tissue is a substance made up of cells that perform a similar function. ... Tumor (American English) or tumour (British English) originally means swelling, and is sometimes still used with that meaning. ... Jump to: navigation, search This stylistic schematic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome (right). ...

Applications

Although SAGE was originally conceived for use in cancer studies, it has been successfully used to describe the transcriptome of other diseases and in a wide variety of organisms. mz-mndjjdnclkn-klnkkdkdkkdk The Transcriptome is the set of all mRNA molecules (or transcripts) in one or a population of biological cells for a given set of environmental circumstances. ...

Comparison to DNA microarrays

The general goal of the technique is similar to the DNA microarray. However, SAGE is a sequence-based sampling technique. Observations are not based on hybridization, which result in more qualitative, analog values. In addition, the mRNA sequences do not need to be known a priori, so genes or gene variants which are not known can be discovered. Microarray experiments are much cheaper to perform, however, so large-scale studies do not typically use SAGE. Jump to: navigation, search A DNA microarray, the different colours indicate relative expression of different genes. ... The interaction of mRNA in a eukaryote cell. ... Jump to: navigation, search A DNA microarray, the different colours indicate relative expression of different genes. ...

Variant Protocols: miRNA cloning

MicroRNAs, or miRNAs for short, are small (~22nt) pieces of RNA which have been found to play a crucial role in gene regulation. One of the most commonly used methods for cloning and identifying miRNAs within a cell or tissue was developed in the Bartel Lab and published in a paper by Lau et al., 2001. Since then, several variant protocols have arisen, but most have the same basic format. The procedure is quite similar to SAGE: The small RNA are isolated, then linkers are added to each, and the RNA is converted to cDNA by RT-PCR. Following this, the linkers, containing internal restriction sites are digested with the appropriate restriction enzyme, and the sticky ends are ligated together into concatemers. Following concatemerization, the fragments are ligated into plasmids and are used to transform bacteria to generate many copies of the plasmid containing the inserts, which may then be sequenced to identify the miRNA present, as well as to observe expression levels of a given miRNA by the number of times it is present in the results, similar to SAGE.

References

• Oncology Centre at Johns Hopkins University
• Velculescu VE, Zhang L, Vogelstein B, and Kinzler KW. 1995. Serial Analysis of Gene Expression. Science 270:484-7 PMID 7570003
• Saha S, Sparks AB, Rago C, Akmaev V, Wang CJ, Vogelstein B, Kinzler KW, Velculescu VE. 2002. Using the transcriptome to annotate the genome. Nat. Biotechnol. 20:508-12 PMID 11981567
• Matsumura H, Ito A, Saitoh H, Winter P, Kahl G, Reuter M, Kruger DH, Terauchi R. 2005. SuperSAGE. Cell Microbiol. 7:11-8 PMID 15617519

External links

• SAGEnet
• SAGE for Beginners