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The aim of this protocol is the digital droplet PCR (ddPCR) quantification of RNA target(s) using primer and probe sets. Reverse transcription and amplification are carried out in a single step, using the One Step RT-ddPCR Advanced kit for probes (Bio-Rad). This protocol is optimised for the analysis of environmental RNA (eRNA) samples (water, sediment, biofilm and soil matrices) and then rare DNA targets. The ddPCR can be performed using a QX600 or a QX200 droplet reader system (Bio-Rad), and this protocol begins after the RNA extraction step and ends with the droplet reading. Multiple eRNA targets can be specifically targeted using multiple primer and probe sets that are compatible. The advantages of using the ddPCR are: the absolute quantification, the sensitivity, the efficiency and the specificity of the method for RNA target quantification.
Vautier et al. (2024) studied this question.