Dataset Information

ARNA-longSAGE: a new approach to generate SAGE libraries from microdissected cells.

ABSTRACT: Large-scale gene expression analyses of microdissected primary tissue are still difficult because generally only a limited amount of mRNA can be obtained from microdissected cells. The introduction of the T7-based RNA amplification technique was an important step to reduce the amount of RNA needed for such analyses. This amplification technique produces amplified antisense RNA (aRNA), which so far has precluded its direct use for serial analysis of gene expression (SAGE) library production. We describe a method, termed 'aRNA-longSAGE', which is the first to allow the direct use of aRNA for standard longSAGE library production. The aRNA-longSAGE protocol was validated by comparing two aRNA-longSAGE libraries with two Micro-longSAGE libraries that were generated from the same RNA preparations of two different cell lines. Using a conservative validation approach, we were able to verify 68% of the differentially expressed genes identified by aRNA-longSAGE. Furthermore, the identification rate of differentially expressed genes was roughly twice as high in our aRNA-longSAGE libraries as in the standard Micro-longSAGE libraries. Using our validated aRNA-longSAGE protocol, we were able to successfully generate longSAGE libraries from as little as 40 ng of total RNA isolated from 2000-3000 microdissected pancreatic ductal epithelial cells or cells from pancreatic intraepithelial neoplasias.

SUBMITTER: Heidenblut AM

PROVIDER: S-EPMC519129 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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Publications

aRNA-longSAGE: a new approach to generate SAGE libraries from microdissected cells.

Heidenblut Anna M AM Lüttges Jutta J Buchholz Malte M Heinitz Christian C Emmersen Jeppe J Nielsen Kåre Lehmann KL Schreiter Pat P Souquet Manfred M Nowacki Sandra S Herbrand Ulrike U Klöppel Günter G Schmiegel Wolff W Gress Thomas T Hahn Stephan A SA

Nucleic acids research 20040915 16

Large-scale gene expression analyses of microdissected primary tissue are still difficult because generally only a limited amount of mRNA can be obtained from microdissected cells. The introduction of the T7-based RNA amplification technique was an important step to reduce the amount of RNA needed for such analyses. This amplification technique produces amplified antisense RNA (aRNA), which so far has precluded its direct use for serial analysis of gene expression (SAGE) library production. We d ...[more]

PMID: 15371555

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Project description:BackgroundSerial Analysis of Gene Expression (SAGE) is a method of large-scale gene expression analysis that has the potential to generate the full list of mRNAs present within a cell population at a given time and their frequency. An essential step in SAGE library analysis is the unambiguous assignment of each 14 bp tag to the transcript from which it was derived. This process, called tag-to-gene mapping, represents a step that has to be improved in the analysis of SAGE libraries. Indeed, the existing web sites providing correspondence between tags and transcripts do not concern all species for which numerous EST and cDNA have already been sequenced.ResultsThis is the reason why we designed and implemented a freely available tool called Identitag for tag identification that can be used in any species for which transcript sequences are available. Identitag is based on a relational database structure in order to allow rapid and easy storage and updating of data and, most importantly, in order to be able to precisely define identification parameters. This structure can be seen like three interconnected modules : the first one stores virtual tags extracted from a given list of transcript sequences, the second stores experimental tags observed in SAGE experiments, and the third allows the annotation of the transcript sequences used for virtual tag extraction. It therefore connects an observed tag to a virtual tag and to the sequence it comes from, and then to its functional annotation when available. Databases made from different species can be connected according to orthology relationship thus allowing the comparison of SAGE libraries between species. We successfully used Identitag to identify tags from our chicken SAGE libraries and for chicken to human SAGE tags interspecies comparison. Identitag sources are freely available on http://pbil.univ-lyon1.fr/software/identitag/ web site.ConclusionsIdentitag is a flexible and powerful tool for tag identification in any single species and for interspecies comparison of SAGE libraries. It opens the way to comparative transcriptomic analysis, an emerging branch of biology.

Dataset Information

ARNA-longSAGE: a new approach to generate SAGE libraries from microdissected cells.

Publications

aRNA-longSAGE: a new approach to generate SAGE libraries from microdissected cells.

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