RT-QuIC Assay for the Detection of Chronic Wasting Disease in Rectal Mucosa of White-Tailed Deer

For each sample, prepare a 2-mL screw cap tube containing 1g of 0.7 mm Zirconia beads (BioSpec 11079107zx) and label with sample/animal ID.

Weigh and add biopsy sample (up to 150mg*) to each tube.

Add 9 volumes 1X PBS 7.4 .

Homogenize using bead beating grinder.

3 cycles of 0h 0m 45s at speed 5.5 with 0h 5m 0s rest On ice between cycles.

Centrifuge at 3000x g.

Collect supernatant*, pooling tubes from larger biopsy samples if necessary.

Prepare 50µL aliquots in 0.2-mL snap cap tubes and reserve remainder in one 2-mL tube. Store at -80°C .

Thaw a 50µL aliquot of 10% homogenate from each sample to be tested.

Sonicate for 0h 0m 30s in a water bath sonicator at a power output of 180-200 W.

Centrifuge ~0h 1m 0s in a benchtop centrifuge at ~2,000 x g (or in a single speed 0.2-mL tube centrifuge) to pellet tissue debris.

Supernatant will be added to the NaPTA reaction in the next step* .

In a 1.5-mL centrifuge tube, combine:

A	B
15 μL	Homogenate supernatant from Step 9
1380 μL	1X PBS pH 7.4
105 μL	4% NaPTA solution

Incubate with gentle rocking or rotation 1h 0m 0s at -80Room temperature .

Pellet by centrifugation 21000x g.

Discard supernatant.*

Resuspend the pellet* in 150µL Resuspension Buffer **.

Substrate Preparation Instructions:

• prepare fresh for each assay run
• rPrP^Csubstrate was shipped as frozen aliquots and filtered before use .

To filter substrate:

1. Thaw aliquot(s) completely at room temperature.
2. Add substrate to 100 kDa centrifugal filter(s).
3. Centrifuge at 3,000 x g for 10 min or until all liquid has passed through the filter
4. Collect filtrate
5. Determine protein concentration*

Prepare RT-QuIC Assay Buffer

• must be prepared fresh for each assay run
• see materials for buffer recipe and Step 15 for substrate handling instructions

Add 98µL RT-QuIC Assay Buffer to each well of a 96-well optical bottom black plate (Thermo Scientific Nunc 265301).

Add 2µL prepared sample from Step 14 to each reaction well.

The following microplate layout is suggested for testing samples in quadruplicate and allows use of a 12-well multichannel pipette for reaction seeding:

A	B
Wells	Sample ID
Column 1 Rows A-D	Sample 1
Column 2 Rows A-D	Sample 2
Column 3 Rows A-D	Sample 3
Column 4 Rows A-D	Sample 4
Column 5 Rows A-D	Sample 5
Column 6 Rows A-D	Sample 6
Column 7 Rows A-D	Sample 7
Column 8 Rows A-D	Sample 8
Column 9 Rows A-D	Sample 9
Column 10 Rows A-D	Sample 10
Column 11 Rows A-D	Sample 11
Column 12 Rows A-D	Sample 12
Column 1 Rows E-H	Sample 13
Column 2 Rows E-H	Sample 14
Column 3 Rows E-H	Sample 15
Column 4 Rows E-H	Sample 16
Column 5 Rows E-H	Sample 17
Column 6 Rows E-H	Sample 18
Column 7 Rows E-H	Sample 19
Column 8 Rows E-H	Sample 20
Column 9 Rows E-H	Sample 21
Column 10 Rows E-H	Sample 22
Column 11 Rows E-H	Sample 23
Column 12 Rows E-H	Sample 24

Seal the plate with film (Thermo Scientific Nunc 232702).

Insert the sealed plate into the microplate reader.

Incubation and Fluorescence Measurement Conditions :

Temperature : 42°C

Shaking : cycles of 700rpm double orbital followed by 0h 1m 0s rest.

Measure : at 0h 43m 0s (or 0h 15m 0s)* intervals: Bottom Read, 20 flashes/well.

Fluorescence: excitation: 450 ± 10 nm, emission: 480 ± 10 nm.

Gain : (manual) 1800.**

Assay length : 85h 0m 0s

For data analysis, we suggest exporting the data in a table format consisting of ThT relative fluorescence units corresponding to each Well/Sample ID at each Measurement Time.

Instructions for data export using BMG MARS software (version ####) are provided below:

Times can be recorded in hour decimal time to facilitate compatibility with downstream calculations:

1. In the MARS software, open the Formats and Settings tab
2. Select the Number Format Settings button
3. Open the Number Formats tab
4. Under Global Time Format Options , select the middle bubble and choose In hours from the drop down menu

To generate an Excel export table:

1. In the MARS software, open the assay file
2. Open the Table View tab
3. Open the drop-down menu Select All Cycles In the top left of the test run window , select the Excel Export button Save the exported file in Excel