20.500.12556/DiRROS-19690 Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system. droplet digital PCR droplet volume DNA quantification optical microscopy imaging true false true Angleški jezik Ni določen Neznano 2024-07-24 03:45:01 2024-07-25 09:48:24 2024-09-10 13:58:42 0000-00-00 00:00:00 2017 0 0 str. 6689-6697 iss. 28 Vol. 409 2017 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2017-09-17 577.2 1618-2642 10.1007/s00216-017-0625-y 4421711 RAZ_Bogozalec_Kosir_Alexandra_2017.pdf RAZ_Bogozalec_Kosir_Alexandra_2017.pdf 1 BEA796FB4D18CADAD0DF006516470C12 9f4dcd56e4eaa3e7c49d457c5666f2cb9c7b53fdb1e205a8165b65450c13d63b fccc98ab-4a49-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27449 https://doi.org/10.1007/s00216-017-0625-y 0 f1b782a4-4a49-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27448 Nacionalni inštitut za biologijo 0 0 0