On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets.
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.
Beer
N Reginald
NR
Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94551, USA. beer2@llnl.gov
Wheeler
Elizabeth K
EK
Lee-Houghton
Lorenna
L
Watkins
Nicholas
N
Nasarabadi
Shanavaz
S
Hebert
Nicole
N
Leung
Patrick
P
Arnold
Don W
DW
Bailey
Christopher G
CG
Colston
Bill W
BW
D001483:Base Sequence; D017931:DNA Primers; D044085:Microfluidics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013050:Spectrometry, Fluorescence
D017931:DNA Primers