Publications

Evaluation of digital PCR for absolute quantification

This poster relates to work carried out under project DD1 - Quality metric toolkit for biomarker discovery and validation (Biomarker metrics) of the NMS Chemical and Biological Metrology programme.

Digital PCR involves diluting and partitioning a sample between many hundreds or thousands of individual PCR reactions such that a single molecule or less on average is present in each reaction. Determination of the number of positive amplifications is indicative of the number of targets present in the sample. As such, digital PCR does not suffer from inaccuracies that often arise from standard real-time PCR approaches at trace levels using calibration curves, for example through extrapolation and it affords the potential for absolute quantification. Recent advances in microfluidics have facilitated the development of digital PCR by combining microfluidics with single molecule, nanolitre volume PCR to increase levels of replication, throughput and cost efficiency.

This poster describes an evaluation of digital PCR for absolute quantification using the Fluidigm BioMark Integrated Microfluidics System as compared to standard real-time PCR using the ABI 7900HT system. The performance of the systems was evaluated using a DNA standard of known molecular weight. The ability of the two platforms to discriminate changes in copy number was assessed using different ratios of RNA standards in a background of human total RNA. Sensitivity was investigated using different starting amounts of the RNA standards.

The poster was presented at the 4^th international qPCR Symposium, held at the University of Munich on 9^th-13^th March.

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