TITLE: RT-QuIC Assay for the Detection of Chronic Wasting Disease in Rectal Mucosa of White-Tailed Deer
AUTHORS: Robert B. Piel, David A. Schneider, Aaron Lomax, Daniel Walsh, Eric M. Nicholson, Tracy A. Nichols, Susan E. Veneziano

[DESCRIPTION]
This protocol details a Real-Time Quaking-Induced Conversion (RT-QuIC) assay to detect the prion-seeding activity associated with Chronic Wasting Disease prions in rectal mucosa samples from white-tailed deer. The authors tested this protocol as part of an agreement between USDA-APHIS, USDA-ARS, and USGS. 


 


[GUIDELINES]
Protocol Notes
SDS is included only in the Resuspension Buffer due to reports that long-term storage in the presence of SDS diminishes the seeding activity of homogenate samples (Caughey Lab, unpublished).
N-2 media supplement is included to buffer adsorption of seeding material to the walls of dilution vessels in diluted homogenate samples. N-2 is not included in initial homogenization buffers as the quantity of total protein in these samples far exceeds the binding capacity of the vessels.



[STEPS]
SECTION: Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
1. For each sample, prepare a 2-mL screw cap tube containing 1 g of 0.7 mm Zirconia beads (BioSpec 11079107zx) and label with sample/animal ID.

SECTION: Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
2. Weigh and add biopsy sample (up to 150 mg*) to each tube.
 

 

SECTION: Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
3. Add 9 volumes 1X PBS pH 7.4 .

 

SECTION: Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
4. Homogenize using bead beating grinder.



SECTION: Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
4.1. 
3 cycles of 45 s at speed 5.5 with 5 min rest on ice between cycles.

SECTION: Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
5. Centrifuge at 3000 x g, 30 s.

SECTION: Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
6. Prepare 50 µL aliquots in 0.2-mL snap cap tubes and reserve remainder in one 2-mL tube. Store at -80 °C .
 


SECTION: Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
7. Thaw a 50 µL aliquot of 10% homogenate from each sample to be tested.
SECTION: Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
8. Sonicate for 30 s in a water bath sonicator at a power output of 180-200 W.


 



SECTION: Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
9. Centrifuge ~1 min in a benchtop centrifuge at ~2,000 x g (or in a single speed 0.2-mL tube centrifuge) to pellet tissue debris.

SECTION: Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
10. In a 1.5-mL centrifuge tube, combine:
      15 μL  supernatant    1380 μL  1X PBS pH 7.4    105 μL  NaPTA solution   4% (w/v) NaPTA, 170 mM MgCl2  
 
SECTION: Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
11. Incubate with gentle rocking or rotation  60 min at -80 Room temperature .

 


SECTION: Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
12. Pellet by centrifugation 21000 x g, 30 min.

SECTION: Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
13. Discard supernatant.*
 

SECTION: Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
14. Resuspend the pellet* in 150 µL Resuspension Buffer**.
 

 



SECTION: RT-QuIC Assay
16. 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

 

SECTION: RT-QuIC Assay
19. Seal the plate with film (Thermo Scientific Nunc 232702).
SECTION: RT-QuIC Assay
21. Incubation and Fluorescence Measurement Conditions:
Temperature: 42 °C
Shaking: cycles of   double orbital followed by 1 min rest.
Measure: at 43 min (or 15 min)* intervals: Bottom Read, 20 flashes/well.
Fluorescence: excitation: 450 ± 10 nm, emission: 480 ± 10 nm.
Gain: (manual) 1800.**
Assay length: 5100 min


 

SECTION: Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
5.1. Collect supernatant*, pooling tubes from larger biopsy samples if necessary.

 

SECTION: RT-QuIC Assay
18. Add 2 µL prepared sample from Step 14 to each reaction well.



SECTION: RT-QuIC Assay
17. Add 98 µLRT-QuIC Assay Buffer to each well of a 96-well optical bottom black plate (Thermo Scientific Nunc 265301). 
SECTION: RT-QuIC Assay
20. Insert the sealed plate into the microplate reader.


 
SECTION: RT-QuIC Assay
18.1. The following microplate layout is suggested for testing samples in quadruplicate and allows use of a 12-well multichannel pipette for reaction seeding:
        Sample   Layout    Wells  Sample    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  
 
SECTION: Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
9.1. Supernatant will be added to the NaPTA reaction in the next step* .

 

SECTION: Export to datafile
22. 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:
SECTION: Export to datafile
22.2. To generate an Excel export table:

In the MARS software, open the assay file
Open the Table View tab
Open the <Select a Cycle group> 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
SECTION: Export to datafile
22.1. Times can be recorded in hour decimal time to facilitate compatibility with downstream calculations:

In the MARS software, open the Formats and Settings tab
Select the Number Format Settings button
Open the Number Formats tab
Under Global Time Format Options, select the middle bubble and choose In hours from the drop down menu
SECTION: rPrPC Substrate Preparation
15. Substrate Preparation Instructions:
prepare fresh for each assay run
rPrPC substrate was shipped as frozen aliquots and filtered before use. 

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