title: Combined RT-qPCR of mRNA and microRNA Targets within One Fluidigm Integrated Fluidic Circuit.
abstract: The ability to profile expression levels of a large number of mRNAs and microRNAs (miRNAs) within the same sample, using a single assay method, would facilitate investigations of miRNA effects on mRNA abundance and streamline biomarker screening across multiple RNA classes. A protocol is described for reverse transcription of long RNA and miRNA targets, followed by preassay amplification of the pooled cDNAs and quantitative PCR (qPCR) detection for a mixed panel of candidate RNA biomarkers. The method provides flexibility for designing custom target panels, is robust over a range of input RNA amounts, and demonstrated a high assay success rate.
authors.affiliation: 1 MSK/dx LLC, Philadelphia, Pennsylvania, USA; and 2 Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Don A
authors.initials: DA
authors.lastname: Baldwin
authors.affiliation: 1 MSK/dx LLC, Philadelphia, Pennsylvania, USA; and 2 Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Annamarie D
authors.initials: AD
authors.lastname: Horan
authors.affiliation: 1 MSK/dx LLC, Philadelphia, Pennsylvania, USA; and 2 Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Hesketh
authors.affiliation: 1 MSK/dx LLC, Philadelphia, Pennsylvania, USA; and 2 Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Samir
authors.initials: S
authors.lastname: Mehta
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000097:blood / Q000235:genetics*; D046210:Microfluidic Analytical Techniques; D012333:RNA, Messenger / Q000097:blood / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015415:Biomarkers; D035683:MicroRNAs; D012333:RNA, Messenger
keywords: high-throughput quantification; long/short RNA