In recent years, several groups have developed PCR chips. Despite the wide spectrum of RNA amplification in molecular diagnosis and research, there is limited work on RT-PCR on chip. Traditionally RT-PCR involves two steps: the RT reaction and PCR amplification. RNA is first reverse transcribed into cDNA using a reverse transcriptase, the resulting cDNA is used as templates for subsequent PCR amplification using primers specific the target gene. RT-PCR can also be carried out as one step RT-PCR in which all reaction components are mixed prior to starting the reactions. One step RT-PCR offers simplicity and convenience and minimizes the possibility for contamination; these characteristics are of particular interest for the development of the proposed abcard. For real-time RT-PCR, the amplification is monitored in real time using fluorescence. Fluorescence signals, proportional to the amount of PCR product can be generated by fluorescent dyes that are specific for double-stranded DNA (dsDNA) or by sequence-specific fluorescent oligonucleotide probes. By using different coloured fluorescent oligonucleotide probes, up to 4 different PCR reactions can be followed simultaneously in a multiplex RT-PCR format. Recently, at DTU-MIC, we have developed a micro-fabricated sample preparation device, a micro-fabricated PCR chip for DNA amplification, and microarrays for identification of Campylobacter species on glass. The small thermal mass of such a PCR microchip enables very fast thermocycling, and we have recently performed 40 PCR cycles within 30 minutes and this approach will be implemented in the new RT-PCR polymeric microchip.

RT-PCR on chip
The right balance between relevant, feasible and efficient research.
IKERLAN-IK4, Technological Research Centre. | J.M. Arizmendiarrieta N2 | 20500 Arrasate-Mondragon | Spain | jmruano@ikerlan.es