Technical concept of a flow-through microreactor for in-situ RT-PCR

This paper presents a novel flow-through reverse transcription-polymerase chain reaction (RT-PCR) microreactor with optimized thermal and fluidic characteristics. It integrates the steps of reverse transcription of the initially applied RNA sample and the amplification of specific DNA fragments of the cDNA formed. The microreactor comprises a heating plate with different temperature zones and an interchangeable fluidic chip with serpentine microchannels. The heating plate provides temperature zones for reverse transcription, hot start activation, denaturation, annealing and extension. The fluidic chip contains a serpentine microchannel with integrated features for segmented sample stream generation and dosing operations, which is guided over the temperature zones according to the thermal protocol of RT-PCR. Reverse transcription is performed in an integrated microchannel section immediately before the amplification of the cDNA's, generated from the primarily applied RNA sample. One feature of this chip system is the generation of a segmented flow for high-throughput analysis of RT-PCR samples. It is shown that RT-PCR reactions can be performed successfully in the microreactor with continuous and segmented flow regimes. The aim of the experiments was the detection of the HPV 16 DNA genome and of viral oncogene transcripts (E6/E7), respectively. Both markers are of importance in medical diagnostics and will be applied in further studies for the identification of oncogene positive cells in cell populations by means of flow-through in-situ RT-PCR.