title: Gene expression analysis by signature pyrosequencing.
abstract: We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.
authors.affiliation: Department of Biotechnology, KTH, Royal Institute of Technology SCFAB, Roslagstullsbacken 21, 106 91 Stockholm, Sweden.
authors.forename: Charlotta
authors.initials: C
authors.lastname: Agaton
authors.forename: Per
authors.initials: P
authors.lastname: Unneberg
authors.forename: Maria
authors.initials: M
authors.lastname: Sievertzon
authors.forename: Anders
authors.initials: A
authors.lastname: Holmberg
authors.forename: Maria
authors.initials: M
authors.lastname: Ehn
authors.forename: Magnus
authors.initials: M
authors.lastname: Larsson
authors.forename: Jacob
authors.initials: J
authors.lastname: Odeberg
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.forename: Joakim
authors.initials: J
authors.lastname: Lundeberg
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012107:Research Design; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C099252:oxidized low density lipoprotein; D013755:Tetradecanoylphorbol Acetate