| Abstract Detail
Conference Wide Nielsen, Kåre L. [1], Laursen Høgh, Annabeth [1], Emmersen, Jeppe [1]. DeepSAGE for gene expression profiling - detection of unknown genes and sensitivity beyond microarrays. SERIAL Analysis of Gene Expression (SAGE) is a digital gene expression profiling method with a dynamical range limited only by the number of sequenced tags. SAGE and LongSAGE are based on the extraction of respectively 14 and 19 nt tags from cDNA. In contrast to microarray, SAGE is an open system that allows the quantification of transcripts that has not been sequenced yet. Therefore, it is ideally suited for comparative gene expression analysis in organisms where a complete genomic sequence is presently unavailable. A drawback of SAGE has been a high cost of experiments compared to DNA microarray analysis (once a comprehensive array has been constructed). Furthermore, SAGE experiments have been quite difficult to perform because of a complex experimental protocol. Recent developments in DNA sequencing, however, facilitate the determination of 25 mio nucleotides of DNA sequence in a single run (www.454.com). We have developed a novel simple protocol for the preparation of LongSAGE ditags, using less than 5% of the material currently use for SAGE library construction. This protocol omits concatenating ditags, their cloning and the subsequent tedious picking of colonies. Using this protocol we have multiplexed two potato LongSAGE samples in a single experiment and obtained 69,994 and 92,815 19 nt tags respectively. The cost per tag was decreased 10 fold, from approximately 0.23 to less than 0.02 €.
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1 - Aalborg University, Department of Biotechnology, Chemistry and Environmental Engineering, Sohngaardsholmsvej 49, Aalborg, 9000, Denmark
Keywords:
expression profiling
Serial analysis of gene expression
sequencing.
Session: Poster-99
Location: Ballroom CD/Monona Terrace
Date: Tuesday, July 25th, 2006
Time: 8:00 AM
Abstract ID:148 |
