title: Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays.
abstract: Gene expression analysis using microarrays of synthetic long oligonucleotides is limited in that it requires substantial amounts of RNA. To obtain these quantities from minute amounts of starting material, protocols were developed that linearly amplify mRNA by cDNA synthesis and in vitro transcription. Since orientation of the product is antisense (aRNA), it is inapplicable for dye-labelling by reverse transcription and hybridization to sense-oriented oligonucleotide arrays. Here, we introduce a novel protocol in which aRNA labelling is achieved by a combination of two reverse and one forward transcription reactions followed by dye-incorporation using Klenow fragment, generating fluorescent antisense cDNA. We demonstrate high fidelity in arrays using up to 10(5)-fold amplification, starting from 2 ng total RNA. The generated data are highly reproducible and maintain relative gene expression levels between samples. These results demonstrate that our protocol describes an efficient and reliable technique to expand the applicability of oligonucleotide arrays to studies where RNA is the limited source material.
authors.affiliation: Division of Molecular Genetics, Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
authors.forename: Joerg
authors.initials: J
authors.lastname: Schlingemann
authors.forename: Olaf
authors.initials: O
authors.lastname: Thuerigen
authors.forename: Carina
authors.initials: C
authors.lastname: Ittrich
authors.forename: Grischa
authors.initials: G
authors.lastname: Toedt
authors.forename: Heidi
authors.initials: H
authors.lastname: Kramer
authors.forename: Meinhard
authors.initials: M
authors.lastname: Hahn
authors.forename: Peter
authors.initials: P
authors.lastname: Lichter
mesh_terms: D016373:DNA, Antisense / Q000737:chemistry; D018076:DNA, Complementary / Q000096:biosynthesis; D005456:Fluorescent Dyes / Q000737:chemistry; D020869:Gene Expression Profiling; D006801:Humans; D016014:Linear Models; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D014158:Transcription, Genetic
chemical_list: D016373:DNA, Antisense; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012333:RNA, Messenger
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