title: On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets.
abstract: The first lab-on-chip system for picoliter droplet generation and RNA isolation, followed by reverse transcription, and PCR amplification with real-time fluorescence detection in the trapped droplets has been developed. The system utilized a shearing T-junction in a fused-silica device to generate a stream of monodisperse picoliter-scale droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing thermal cycling for reverse transcription and subsequent PCR amplification without droplet motion. This combination of the established real-time reverse transcription-PCR assay with digital microfluidics is ideal for isolating single-copy RNA and virions from a complex environment and will be useful in viral discovery and gene-profiling applications.
authors.affiliation: Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94551, USA. beer2@llnl.gov
authors.forename: N Reginald
authors.initials: NR
authors.lastname: Beer
authors.forename: Elizabeth K
authors.initials: EK
authors.lastname: Wheeler
authors.forename: Lorenna
authors.initials: L
authors.lastname: Lee-Houghton
authors.forename: Nicholas
authors.initials: N
authors.lastname: Watkins
authors.forename: Shanavaz
authors.initials: S
authors.lastname: Nasarabadi
authors.forename: Nicole
authors.initials: N
authors.lastname: Hebert
authors.forename: Patrick
authors.initials: P
authors.lastname: Leung
authors.forename: Don W
authors.initials: DW
authors.lastname: Arnold
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Bailey
authors.forename: Bill W
authors.initials: BW
authors.lastname: Colston
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D044085:Microfluidics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013050:Spectrometry, Fluorescence
chemical_list: D017931:DNA Primers