Protein Misfolding Cyclic Amplification of Prions

1. Preparing the Equipment

1. Use a Misonix 3000 or Misonix 4000 sonicator (Farmingdale, NY) connected to a Thermo Electron Neslab EX-7 water bath (Newington, NH) to keep a constant temperature of 37 °C. Sonicate the samples in 200 μl thin-walled PCR tube strips with domed caps obtained from Thermo Scientific (Waltham, MA).
2. A brand new sonicator requires a “break-in” period of continuous operation⁹. A two-month break-in period consists of a 40-sec sonication burst and 10-minute incubation cycles with the amplitude level set to 10. One should observe erosion throughout the surface of the titanium microplate horn (Figure 1, panel B). The circulating water will look white turbid due to erosion of the titanium microplate horn. Replace this water daily.
3. The amplification of PrP^Sc will vary throughout the microplate horn. The best PrP^Sc amplification efficiency can be obtained within a radius of 5 cm from the center of the microplate horn (See Figure 1, panel C).
4. One round of PMCA consists 144 cycles. Each cycle consists of 5 sec sonication and 10 min incubation. This will roughly entail to 24 hr per round of PMCA.
5. The power output of sonication, displayed on the sonicator’s control panel, varies with the number of PCR tubes present in the sonicator’s rack and the temperature of the circulating water from the water bath. Ensure that the water is equilibrated at 37 °C, and do not fill more than 30% of the sonicator’s tube rack (Figure 1, panel C). Spread the PCR tubes through the microplate horn. Have at least one row of empty space between PCR tube strips and allow no more than four tubes connected together per strip (Figure 1, panel C).
6. The strength of sonication is critical for a successful prion strain amplification. While sonicating, adjust the amplitude to have a power output ranging between 155 ~ 170 watts. In our laboratory the power level is set to six and seven for the Misonix 3000 and Misonix 4000, respectively.
7. Use distilled water in the water bath to avoid accumulating hard water stains. Replace this water weekly. During the incubation and sonication cycles, water condensation will occur inside the microplate horn lid. To avoid condensation, attach a small aquarium heat pad to the top of the sonicator’s enclosure box as shown in Figure 1, panel A.

2. Preparing the Samples

1. All procedures involving animals were approved by the Creighton University Institutional Animal Care and Use Committee and comply with the Guide for the Care and Use of Laboratory Animals. Before any tissue collection, the animal must be anesthetized and transcardially perfused using ice-cold phosphate buffered saline solution with 5 mM EDTA at pH 7.4 to remove blood from the brain and avoid the inhibition of PMCA reaction by blood⁷. Collect animal tissues rapidly using aseptic technique and have dedicated dissection tools for each prion strain. After dissection, place the tissue in a 1.5 ml microcentrifuge tube, flash freeze it on dry ice, and store it at -80 °C until homogenization.
2. To prepare the PMCA substrate (PrP^C) solution, homogenize an uninfected hamster brain (UN BH) to a 10% weight/volume (w/v) solution with ice cold conversion buffer (see part 3) using a Tenbroeck tissue grinder. While homogenizing, be careful not to get air inside the grinder to avoid excessive foaming of the solution. Clarify the solution by centrifuging at 500 x g for 30 sec and aliquot the supernatant in 1.5 ml microcentrifuge tubes. Store at -80 °C until further use.
3. To prepare the PMCA seed (PrP^Sc) solution, homogenize an infectious material (brain, spleen, lymph nodes, other tissues or contaminated soil) to a 10% w/v solution with ice cold water and aliquot the solution in 0.6 ml microcentrifuge tubes. Store at -80 °C until further use.

3. Preparing Conversion Buffer

1. Weigh 0.5093 g of Triton X-100 in a 50 ml volumetric flask (result is a 1% final concentration of Triton X-100) (Sigma-Aldrich GmbH; Steinheim, Germany).
2. Add one Complete Protease Inhibitor Tablet (Roche Diagnostics GmbH; Mannheim, Germany).
3. Add 0.0931 g of EDTA (this will result in a 5 mM final concentration of EDTA) (Mallinckrodt Baker Inc.; Phillipsburg, NJ).
4. Adjust the pH to 7.4 with 1N NaOH.
5. Adjust volume to 50 ml with Dulbecco’s Phosphate Buffered Saline (DPBS) (Mediatech Inc.; Manassas, VA).
6. Filter the solution through a 0.45 μm nitrocellulose membrane with a vacuum filtration apparatus. The conversion buffer can be stored at 4 °C for no more than one month.

4. Amplification of Prion Strains Using PMCA

1. Dilute the PMCA seed into the PMCA substrate at a ratio of 1:20 (i.e. mix 5 μl of the PMCA seed and 95 μl of the PMCA substrate) into a PCR tube. Remove and store a 15 μl aliquot at -80 °C for unsonicated control; this unsonicated control will be used to quantify the PrP^Sc amplification via proteinase K (PK) digestion followed by Western blot (WB) analysis. An additional unsonicated control could be generated by placing a second 15 μl aliquot at 37 °C for the duration of the PMCA reaction. A minimum of three repetitions is required for statistical purposes.
2. Subject the remainder of the sample (70 μl) to one round of PMCA as depicted on Figure 1, Panel A. Shake tubes every 5 hr to avoid water condensation on the domed cap of the PCR tube.
3. The sample’s tubes should be spun down and gently vortexed (being careful that the solution do not go into the domed cap) before opening after any procedure to ensure homogeneity and avoid potential contamination.
4. For serial PMCA (sPMCA) of short incubation period strains (i.e. HY TME, HaCWD, or 263K), dilute the sonicated material at a ratio of 1:20 by transferring 5 μl of the sonicated sample from round one into 95 μl of fresh uninfected brain homogenate (Figure 2, Panel B).
5. For sPMCA of long incubation period strains (i.e. DY TME, 139H, 22CH, 22AH, or ME7H), dilute the sonicated material at a ratio of 1:1 by transferring 50 μl of the sonicated sample from round one into 50 μl of fresh uninfected brain homogenate (Figure 2, Panel B).
6. Remove two 15 μl aliquots from the previously diluted sonicated mixture (from steps 4.4 or 4.5) and store one of them at -80 °C and the other at 37 °C (unsonicated control solutions for PMCA round two). Subject the remnants to a PMCA round two.
7. This procedure can be repeated indefinitely. In our laboratory, we have performed up to fifteen PMCA rounds, with the strain properties remaining unchanged.
8. PK digest the samples. For a typical WB, mix 5 μl of PK 80 μg/ml to 5 μl of sample (this solution will have a final PK concentration of 40 μg/ml) and incubate at 37 °C for one hour. Add 10 μl of gel loading buffer. Boil this solution for 10 min. Let the solution cool down and load 10 μl into the gel.
9. To calculate the success of amplification perform a WB analysis on the PK-digested samples to estimate the amount of amplified PrP^Sc by comparing them to either, the unsonicated controls^20,21 Figure 2, Panel C or to known amounts of PK-undigested recombinant PrP.

5. Avoiding Cross-contamination

Critical steps have to be taken to minimize cross-contamination and the occurrence of false positives due to de novo formation of protease resistant products.

1. Use a dedicated set of dissection tools (i.e. forceps, tweezers, scissors) for sample collection for each prion strain.
2. Before homogenizing uninfected brains to be used as a PMCA substrate, wipe the pipettes with 1% bleach. Soak the sonicator’s PCR tube rack, tissue homogenizers, and PCR tube storage racks with 1% bleach for 10 min and rinse thoroughly with distilled water.
3. Cover the work area with a fresh Versi-Dry Lab Soaker every time a new sample is to be homogenized, a new PMCA experiment is to be prepared, or when aliquoting between sPMCA rounds.
4. Use new gloves each time new samples are handled (especially when transferring aliquots between sPMCA rounds).
5. Use short sonication bursts for each PMCA cycle. Cycles of 5 sec sonication combined with 10 min incubation is sufficient to amplify prions without generating de novo PrP^{Sc 1,20,21,23,24} . As other laboratories have shown, increasing the sonication time, increasing the sonication amplitude, and extending the number of PMCA cycles increases the probability for de novo PrP^Sc formation².