Reaction-mapped quantitative multiplexed polymerase chain reaction on a microfluidic device.
We have applied multiple-time-point reaction mapping to generate high-dynamic-range quantitative data from PCR multiplexes. The approach measures, then compensates, numerous PCR slope nonidealities across the multiplex without prejudice. A multilane microelectophoresis device with a novel scanning detector that reports redundantly over more than six decades in signal strength was used to collect data with multiple readings for each amplification point and with double internal calibration (lane standards and gene standards). We investigated scaling properties and sensitivity for readout of 12plex PCR reactions. The sensitive detection, stemming from confocal optics, allowed reduction of the PCR cycle number by approximately five cycles compared to commercial fluorometric readout. This increased sensitivity appears to allow quantitative PCR over a dynamic range of >9 log2 abundance ratio in multiplex reactions exceeding 20plexes. We argue that the combination of mapping, multiplexing, and an internal standard, improves the per-well efficiency of quantitative expression analysis by a factor of 50-100 relative to fluorometric qPCR readout. Therefore, the approach is attractive for analysis of large gene networks at reduced cost.
Ueberfeld
Jörn
J
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142, USA.
McKenna
Brian
B
Rubin-Bejerano
Ifat
I
Verstrepen
Kevin
K
Ehrlich
Daniel J
DJ
D002176:Candida albicans / Q000235:genetics / Q000302:isolation & purification; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D009504:Neutrophils / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012331:RNA, Fungal / Q000032:analysis; D015203:Reproducibility of Results
D012331:RNA, Fungal