Table 1.
PMCA and RT-QuIC assays protocols.
| Assay | Sample | α-syn concentration (mg/ml) | Buffer | Sonication/shaking | Temperature (°C) | Beadsa | Sample volume/final volume (μl) | Sample readout | Number of samples (cases/controls) | Sensitivity (%) | Specificity (%) | Detection limitsb | References |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| RT-QuIC | BH; CSF | 0.1 | 40 mM phosphate (pH 8.0), 170 mM NaCl, 10 μM ThT, 0.0015% SDS (only for CSF) |
1 min (400 rpm) 1 min rest (Sh) |
42 | 6 beads (0.8 mm) | BH 2/100; CSF 15/100 |
ThT | 29/31 | 93 | 100 | BH 10−6; CSF 0.2 μl; α-syn fibrils 100 ag | (7) |
| RT-QuIC | BH | 0.1–0.15 | 50 mM HEPES (pH 7.5), 10 μM ThT |
40 s (432 rpm), 2 min rest (Sh) |
40 | – | BH (diluted) 5/100 | ThT | 13/2 | – | 100 | BH 5 × 10−6 | (6) |
| RT-QuIC | BH; CSF | 0.1 | 100 mM phosphate (pH 8.2), 10 μM ThT |
1 min (200 rpm), 14 min rest (Sh) |
30 | 37 ± 3 mg (0.5 mm) |
BH (1:20,000) 2/100; CSF 5–15/100 |
ThT | BH, 15/3; CSF, 102/35 |
92 DLB, 95 PD |
100 | – | (5) |
| PMCA | CSF | 0.1 or 1 | PBS (pH 7.4) or 100 mM PIPES (pH 6.5), 500 mM NaCl, 5 μM ThT |
1 min (500 rpm), 29 min rest (Sh) |
37 | – | CSF 40/200 |
ThT | 96/97 | 100 DLB, 88.5 PD, 80 MSA |
94–96.9 | 150 amol | (4) |
| PMCA | BH | 0.723 | 10 mM Tris (pH 7.5), 150 mM NaCl |
10 s every 29 min 50 s (So) |
37 | 10 beads (1 mm) | BH 1% | ThT | 1/1 | – | – | 100 amol | (76) |
| PMCA | – | 0.38 or 1.28 | 150 mM NaCl, 1% triton X-100, complete protease inhibitors in PBS |
20 s every 30 min (So) |
37 | 37 ± 3 mg (1 mm) |
200 | ThT; WB | – | – | – | – | (80) |
| PMCA | – | 0.28 | 150 mM NaCl, 1% triton X-100 |
20 s every 29 min 40 s (So) |
37 | – | 100 | ThT; WB | – | – | – | – | (85) |
| PMCA | – | 4.3 | Physiological buffer (pH 7.4) |
30 s every 10 min (x6) (So); 10 min (300 rpm, x6), 10 h constant, 10 min (x6) (Sh) |
37 | – | 300 | WB | – | – | – | – | (86) |
BH, brain homogenates; CSF, cerebrospinal fluid; SDS, sodium dodecyl sulfate; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); ThT, Thioflavin T; PIPES, Piperazine-N,N'-bis(ethansulfonic acid); PBS, phosphate-buffered saline; So, sonication; Sh, shaking; WB, western blot; DLB, dementia with Lewy bodies; PD, Parkinson's disease; MSA, multiple system atrophy; ag, attograms; amol, attomoles.
a
Number/mg of zirconia/silica beads per reaction.
b
Detection limits as reported in the original papers.