Serial Analysis of Gene Expression
Encyclopedia
Serial analysis of gene expression
(SAGE) is a technique used by molecular biologist
s to produce a snapshot of the messenger RNA
population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu
at the Oncology Center of Johns Hopkins University
and published in 1995. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGE and the most recent SuperSAGE
. Many of these have improved the technique with the capture of longer tags, enabling more confident identification of a source gene.
A more in-depth, technical explanation of the technique is available here.
s a researcher can usually determine, with some confidence, the original mRNA (and therefore which gene
) the tag was extracted from.
Statistical methods can be applied to tag and count lists from different samples in order to determine which gene
s are more highly expressed. For example, a normal tissue
sample can be compared against a corresponding tumour to determine which gene
s tend to be more (or less) active.
of other diseases and in a wide variety of organisms.
. However, SAGE sampling is based on sequencing mRNA output, not on hybridization of mRNA output to probes, so transcription levels are measured more quantitativly than by microarray. 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, so large-scale studies do not typically use SAGE. Quantifying gene expressions is more exact in SAGE because it involves directly counting the number of transcripts whereas spot intensities in microarrays fall in non-discrete gradients and are prone to background noise.
, or miRNAs for short, are small (~22nt) segments 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 concatamers. Following concatenation, the fragments are ligated into plasmids and are used to transform bacteria to generate many copies of the plasmid containing the inserts. Those may then be sequenced to identify the miRNA present, as well as analysing expression levels of a given miRNA by counting the number of times it is present, similar to SAGE.
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
(SAGE) is a technique used by molecular biologist
Biologist
A biologist is a scientist devoted to and producing results in biology through the study of life. Typically biologists study organisms and their relationship to their environment. Biologists involved in basic research attempt to discover underlying mechanisms that govern how organisms work...
s to produce a snapshot of the messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu
Victor Velculescu
Victor E. Velculescu is a Romanian/American doctor and scientist. He is Associate Professor of Oncology and Co-Director of Cancer Biology at the Sidney Kimmel Cancer Center at The Johns Hopkins University....
at the Oncology Center of Johns Hopkins University
Johns Hopkins University
The Johns Hopkins University, commonly referred to as Johns Hopkins, JHU, or simply Hopkins, is a private research university based in Baltimore, Maryland, United States...
and published in 1995. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGE and the most recent SuperSAGE
SuperSAGE
SuperSAGE is the most advanced derivate of the serial analysis of gene expression technology for the analysis of expressed genes in eukaryotic organisms . Like in SAGE, a specific tag from each transcribed gene is recovered...
. Many of these have improved the technique with the capture of longer tags, enabling more confident identification of a source gene.
Overview
Briefly, SAGE experiments proceed as follows:- Isolate the mRNA of an input sample (e.g. a tumour).
- Extract a small chunk of sequence from a defined position of each mRNA molecule.
- Link these small pieces of sequence together to form a long chain (or concatemer).
- Clone these chains into a vectorVector (molecular biology)In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
which can be taken up by bacteria. - Sequence these chains using modern high-throughput DNA sequencerDNA sequencerA DNA sequencer is a scientific instrument used to automate the DNA sequencing process. It can be also considered an optical instrument as it generally analyses light signals originating from fluorochromes attached to nucleotides....
s. - Process this data with a computer to count the small sequence tags.
A more in-depth, technical explanation of the technique is available here.
Analysis
The output of SAGE is a list of short sequence tags and the number of times it is observed. Using sequence databaseSequence database
In the field of bioinformatics, a sequence database is a large collection of computerized nucleic acid sequences, protein sequences, or other sequences stored on a computer...
s a researcher can usually determine, with some confidence, the original mRNA (and therefore which gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
) the tag was extracted from.
Statistical methods can be applied to tag and count lists from different samples in order to determine which gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s are more highly expressed. For example, a normal tissue
Biological tissue
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
sample can be compared against a corresponding tumour to determine which gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s tend to be more (or less) active.
Applications
Although SAGE was originally conceived for use in cancer studies, it has been successfully used to describe the transcriptomeTranscriptome
The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced in one or a population of cells.-Scope:...
of other diseases and in a wide variety of organisms.
Comparison to DNA microarrays
The general goal of the technique is similar to the DNA microarrayDNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
. However, SAGE sampling is based on sequencing mRNA output, not on hybridization of mRNA output to probes, so transcription levels are measured more quantitativly than by microarray. 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
DNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
experiments are much cheaper to perform, so large-scale studies do not typically use SAGE. Quantifying gene expressions is more exact in SAGE because it involves directly counting the number of transcripts whereas spot intensities in microarrays fall in non-discrete gradients and are prone to background noise.
Variant Protocols: miRNA cloning
MicroRNAsMirna
Mirna may refer to:geographical entities* Mirna , a river in Istria, Croatia* Mirna , a river in Slovenia, tributary of the river Sava* Mirna , a settlement in the municipality of Mirna in Southeastern Sloveniapeople...
, or miRNAs for short, are small (~22nt) segments 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
Reverse transcription polymerase chain reaction
Reverse transcription polymerase chain reaction is a variant of polymerase chain reaction , a laboratory technique commonly used in molecular biology to generate many copies of a DNA sequence, a process termed "amplification"...
. Following this, the linkers, containing internal restriction sites, are digested with the appropriate restriction enzyme and the sticky ends are ligated together into concatamers. Following concatenation, the fragments are ligated into plasmids and are used to transform bacteria to generate many copies of the plasmid containing the inserts. Those may then be sequenced to identify the miRNA present, as well as analysing expression levels of a given miRNA by counting the number of times it is present, similar to SAGE.