Aug 26, 2022
RT-QuIC alpha-synuclein
- 1University Medical Center Goettingen
Protocol Citation: Mary Xylaki 2022. RT-QuIC alpha-synuclein . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7yzb3gwz/v1
Manuscript citation:
Russo MJ, Orru CD, Concha-Marambio L, Giaisi S, Groveman BR, Farris CM, et al. High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson’s disease. Acta Neuropathol Commun. BioMed Central; 2021;9:1–13. Concha-Marambio L, Shahnawaz M, Soto C. Detection of Misfolded α-Synuclein Aggregates in Cerebrospinal Fluid by the Protein Misfolding Cyclic Amplification Platform. 2019. p. 35–44.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69179
Keywords: ASAPCRN
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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.
Materials
- BMG Technologies Omega FLUOstar Reader or similar fluorometer
- regular lab equipment (balance, pHmeter, pipetts, tips, tubes)
- COSTAR 96-well ELISA plates (Corning, cat# 3916)
- MicroAmp Film (Applied Biosystems, cat# 4311971)
- Si3N4 beads 2.38 mm
- molecular biology grade nuclease free water
- 0.5 M PIPES pH 6.5
- 5 M NaCl
- Thioflavin T dissolven in water
- recombinant C-terminal His-tag alpha synuclein
- 1% BSA
- 5 N NaOH and 1N HCl for pH adjustment
Bead preparation
Bead preparation
Beads are blocked with 1% BSA in 100mM PIPES for 1 hr and washed twice with PIPES.
Preparation of Reaction buffer
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
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
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.