title: Rapid, quantitative, reverse transcription PCR in a polymer microfluidic chip.
abstract: Quantitative PCR (qPCR) techniques have become invaluable, high-throughput tools to study gene expression. However, the need to measure gene expression patterns quickly and affordably, useful for applications such as stem cell biomanufacturing requiring real-time observation and control, has not been adequately met by rapid qPCR instrumentation to date. We report a reverse transcription, microfluidic qPCR system and its application to DNA and RNA amplification measurement. In the system, an environmental control fixture provides mechanical and thermal repeatability for an infrared laser to achieve both accurate and precise open-loop temperature control of 1 μl reaction volumes in a low-cost polymer microfluidic chip with concurrent fluorescence imaging. We have used this system to amplify serial dilutions of λ-phage DNA (10(5)-10(7) starting copies) and RNA transcripts from the GAPDH housekeeping gene (5.45 ng total mouse embryonic stem cell RNA) and measured associated standard curves, efficiency (57%), repeatability (~1 cycle threshold), melting curves, and specificity. This microfluidic qRT-PCR system offers a practical approach to rapid analysis (~1 h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easy setup.
authors: GW Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
authors: D Curtis
authors: DC
authors: Saunders
authors: Gregory L
authors: GL
authors: Holst
authors: Christopher R
authors: CR
authors: Phaneuf
authors: Nikita
authors: N
authors: Pak
authors: Matthew
authors: M
authors: Marchese
authors: Nicholas
authors: N
authors: Sondej
authors: Michael
authors: M
authors: McKinnon
authors: Craig R
authors: CR
authors: Forest
mesh_terms: D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D004867:Equipment Design; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000191:economics / Q000295:instrumentation*; D012313:RNA / Q000032:analysis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000295:instrumentation; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000295:instrumentation*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D004279:DNA, Viral; D012313:RNA; C546243:GAPDH2 protein, mouse; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)