Table 4. Troubleshooting guide for RT-QuIC assay issues.

Problem	Potential causes	Solutions
Delayed or no ThT fluorescence increase	(1) Insufficient seeding activity in TCA-enriched samples (2) Suboptimal SDS or ThT concentration	(1) Increase seed concentration to 2 μg/mL or extend reaction time to 72 h (2) Optimize SDS concentration (0.001%–0.003%) to balance fibril formation
High background fluorescence in negative controls	(1) Contamination from reagents or equipment (2) Premature fibrillization due to temperature fluctuations	(1) Use fresh, sterile-filtered buffers and UV-treated consumables (2) Pre-chill plates on ice and maintain 37°C during incubation
Inconsistent fibril formation	(1) Variability in recombinant α-Syn batch (2) Uneven plate shaking	(1) Use monomeric, endotoxin-free α-Syn validated by SEC (2) Ensure consistent 600 r/min shaking during RT-QuIC