title: High throughput microRNA profiling: optimized multiplex qRT-PCR at nanoliter scale on the fluidigm dynamic arrayTM IFCs.
abstract: The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.
authors.affiliation: The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, USA.
authors.forename: Felix
authors.initials: F
authors.lastname: Moltzahn
authors.forename: Nathan
authors.initials: N
authors.lastname: Hunkapiller
authors.forename: Alain A
authors.initials: AA
authors.lastname: Mir
authors.forename: Tal
authors.initials: T
authors.lastname: Imbar
authors.forename: Robert
authors.initials: R
authors.lastname: Blelloch
mesh_terms: D000818:Animals; D053595:Embryonic Stem Cells / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D046210:Microfluidic Analytical Techniques; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*
chemical_list: D035683:MicroRNAs