Better accuracy of nucleic acids measurements to improve digital PCR instruments

Nano-liter droplets encapsulating DNA molecules detected by digital PCR © EU, 2015

In close collaboration with a research group from the Australian National Measurement Institute in Pymble, JRC scientists have improved the measurement of nucleic acids by using a JRC certified reference material. This is important for digital polymerase chain reaction (dPCR) instrument developers.

During the last 25 years, quantitative PCR (qPCR) has been the method of choice to determine nucleic acid molecules in many applications of green and red biotechnologies. qPCR has been applied to gene expression quantification, forensic DNA quantification, clinical, veterinary and  virology diagnostics. The necessity to calibrate qPCR with appropriate and well-characterised calibrants remains a major limitation of qPCR as such calibrant are not always appropriate or available.

Since 2009, the JRC-Institute for Reference Materials (IRMM) is collaborating  with the Australian National Institute of Measurement and was the first to identify the experimental parameters that affect the reliability of dPCR results. At that time, dPCR experiments were performed in microfluidic chips. Since then, newer dPCR instruments came on the market in which the DNA solution is portioned in thousands of droplets.
Knowing the precise volume (in the range of one nano-liter) of such droplets is crucial as it is needed to calculate the DNA concentration. Because the JRC-IRMM had developed a certified reference material ERM-AD623 which provided anchor points for DNA quantification, it could be demonstrated that the average volume of the droplets generated by a droplet dPCR apparatus was incorrectly estimated and led to an underestimation of the reported DNA concentration by 8 %.

This finding was acknowledged by a life science company producing and distributing those dPCR apparatus worldwide. The manufacturer decided to release a new software version that takes into account the volume of the droplets as measured by the JRC-IRMM. It could be demonstrated that dPCR results generated by chip- and droplet-based dPCR apparatus agree when the correct droplet volume is taken into account.  This is a step forward to establish dPCR as a reliable and accurate diagnostic tool.

Read more in:
S. Bhat et al.: “Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number“,  Anal Bioanal Chem. 394 (2009) 457-67, doi: 10.1007/s00216-009-2729-5

P. Corbisier et al.: “DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials“,  Anal Bioanal Chem 407 (2015) 1831–1840, doi: 10.1007/s00216-015-8458-z