Rapid, quantitative, reverse transcription PCR in a polymer microfluidicchip

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 (105–107 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 easysetup.

A user-friendly instrument for microfluidic quantitative PCR and RT-PCR is reported.
► Plastic, disposable microchips were used to contain 1 μl reaction volumes.
► Precision mechanical alignment and thermal stability allow repeatable open-loop control.
► Quantitative PCR and RT-PCR with viral DNA and GAPDH RNA were demonstrated.
► Electrophoretic end-point detection and melting curve analysis confirmed amplifications.