Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We developed an Excel based ‘experience matrix’ that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Optimized assays in return minimize the impact of rain on ddPCR analysis. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Signals of such droplets can hinder analysis and the correct setting of a threshold. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called ‘rain’.
This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. No., catalogue number cp/cp, (gene) copy per (gene) copy dPCR, digital PCR ddPCR, droplet digital PCR DNA, deoxyribonucleic acid EC, European Commission ERM, Certified European Reference Material EU, European Union EURL-GMFF, European Reference Laboratory for GM Food and Feed fluorescein, FAM,F gDNA, genomic DNA GM, genetically modified GMO, genetically modified organism HEX,H, hexachlorfluorescein L, liter Lec, lectin gene of soy MeanSignal, mean fluorescence signal value MIQE, minimal information for publication of quantitative digital PCR experiments MRPL, minimum required performance limit MS, Microsoft MWG, Eurofins-MWG qPCR, (quantitative) real-time PCR PCR, polymerase chain reaction SD, standard deviation (of fluorescence signals) TAMRA,T, tetramethylrhodamin Tech, technician TIB, TIB Molbiol VBA, visual basic for applications VIC,V, fluorescent dye (LifeTechnologies)ĭigital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Abbreviations: ABI, LifeTechnologies (formerly AppliedBiosystems) Bio, DNA Technology/Biosearch Technologies Cat.
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