Linearity, limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ)

We performed two parallel dilution experiments to assess the linearity of the miR371 assay in RT-qPCR with pre-amplification and ddPCR without pre-amplification. First, we performed a two-fold serial dilution of RNA from a patient sample with known positive miR371 expression in nuclease-free water as described by Hindson et al.²⁸. Three parallel dilution series were made, cDNA was synthesised and thereafter analysed by qPCR with pre-amplification and ddPCR (without pre-amplification). Second, for further assessment of the performance and precision at low concentrations we performed an additional dilution series at the lower range. cDNA was diluted two-fold and ten qPCR and ddPCR reactions were carried out for each dilution. In order to assess the reproducibility of the miR371 assay towards the LOD, we selected 13 samples with negative or low miR371 levels and extracted RNA on a different day and measured miR371 a second time. Measurements by ddPCR and qPCR were performed from the same cDNA, and the results from the two runs (day 1 vs. day 2) were compared. The repeatability of the protocol from RNA extraction through qPCR with pre-amplification and ddPCR was tested by repeated measurement of miR371 in samples from three patients with different levels of miR371 expression.

LOB and LOD for RT-ddPCR were established according to Armbruster et al.⁵³.

LOQ was defined as the lowest concentration that could be detected with CV ≤ 25%⁵⁴.