Establishing the quantitative RT-QuIC (qRT-QuIC). (a) Schematic illustration of qRT-QuIC. The PrP27-30 propagating duration (hour) required to reach the threshold which was at least 3 times the starting fluorescence was set as the independent variable (x), the correlated seeded amount of PrP27-30 was the dependent variable (y). (b) Different amounts of purified PrP27-30 (10-10 to 10-16 g with serial 100.5-fold dilution) were seeded into RT-QuIC reactions using mouse rPrPsen as the substrate. The RT-QuIC process was followed from 0 to 90 h by showing the number of hours required to reach the threshold (indicated by black arrows and intersecting lines). Purified PrPC with identical amounts was seeded in independent RT-QuIC reactions as control. Seeded amounts of both PrP27-30 and PrPC are indicated on the top right; see also Additional file 1: Figures S2 and S3. (c) The results from five repeats of RT-QuIC seeded by PrP27-30 were provided to yield standard calibration curves and formulas for quantification. This relates QuIC time necessary to reach the threshold to the amount of seeded PrP27-30. The mean and s.e.m. are shown (n = 5).