RT-QuIC alpha-synuclein

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Abstract

This protocol is for the detection of prionoid alpha-synuclein forms in human cerebrospinal fluid using the Real Time Quacking-Induced Conversion method (RT-QuIC). The protocol is adapted from Marco J. Russo, Christina D. Orru, Luis Concha‑Marambio, Simone Giaisi et al., 2021 (doi:10.1186/s40478-021-01282-8) and Concha-Marambio et al., 2019 (doi:10.1007/978-1-4939-9124-2_4).

This assay is for research use only and not diagnostics.

Steps

Bead preparation

Beads are blocked with 1% BSA in 100mM PIPES for 1 hr and washed twice with PIPES.

Preparation of Reaction buffer

Reaction mixture is prepared calculating 200 μl per well with the following final concentrations:

0.3 mg/mL recombinant alpha-synuclein
0.5 M NaCl
100 mM PIPES buffer
5 μM ThT

Seeting up the assay

One bead is placed in each well of the assay plate. 160 μl reaction buffer and 40 μl CSF are carefully pipetted in each well. Samples are assessed in triplicates.

Plate is covered with the film and creases are removed manually.

Plate is placed in the plate reader and incubated for 240 hrs at 37 °C in cycles of 1 min shaking at 500rpm, 29 mins incubation and fluorescence measurements are taken after every incubation cycle at 440ex/490emm.

Data analysis

A sample is considered positive when it crosses a fluorescence threshold established at 3 standard deviations above baseline. 3/3 wells are positive and negative when 1/3 samples are positive while 2/3 is considered as incoclusive.

Relative fluorescence units measured at 490 are plotted versus the time in hours and should present a classical exponantial curve with lag phase and plateau. Kinetics parameters obtained from the curves include maximum fluorescence, time to reach 50% of maximal fluoresence and time to reach the established threshold.