Rapid, quantitative, reverse transcription PCR in a polymer microfluidicchip

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 1μl 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 (105–107 starting copies) and RNA transcripts from the GAPDH housekeeping gene (5.45ng 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 (∼1h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easysetup.