title: Universal nucleic acid sequence-based amplification for simultaneous amplification of messengerRNAs and microRNAs.
abstract: A universal NASBA assay is presented for simultaneous amplification of multiple microRNA (miRNA) and messengerRNA (mRNA) sequences. First, miRNA and mRNA sequences are reverse transcribed using tailed reverse transcription primer pairs containing a gene-specific and an non-specific region. For reverse transcription of small miRNA molecules a non-specific region is incorporated into a structured stem-loop reverse transcription primer. Second, a universal NASBA primer pair that recognizes the tagged cDNA molecules enables a simultaneous, transcription-based amplification reaction (NASBA) of all different cDNA molecules in one reaction. The NASBA products (RNA copies) are detected by gene-specific DNA probes immobilized on a biochip. By using the multiplex reverse transcription combined with the universal NASBA amplification up to 14 different mRNA and miRNA sequences can be specifically amplified and detected in parallel. In comparison with standard multiplex NASBA assays this approach strongly enhances the multiplex capacity of NASBA-based amplification reactions. Furthermore simultaneous assaying of different RNA classes can be achieved that might be beneficial for studying miRNA-based regulation of gene expression or for RNA-based tumor diagnostics.
authors.affiliation: Laboratory for Chemistry and Physics of Interfaces, Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Köhler-Allee 103, D-79110 Freiburg, Germany. andreas.mader@imtek.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Mader
authors.forename: Ulrike
authors.initials: U
authors.lastname: Riehle
authors.forename: Thomas
authors.initials: T
authors.lastname: Brandstetter
authors.forename: Elmar
authors.initials: E
authors.lastname: Stickeler
authors.forename: Juergen
authors.initials: J
authors.lastname: Ruehe
mesh_terms: D001483:Base Sequence; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D021161:Self-Sustained Sequence Replication / Q000295:instrumentation
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger