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Updated: Dec 26, 2025
Protein Misfolding Cyclic Amplification of Prions
Published on: November 7, 2012
Amplification Techniques for the Detection of Misfolded Prion Proteins in Experimental and Clinical Samples
1College of Graduate Studies, Department of Microbiology and Immunology, Midwestern University, Glendale, Arizona.
Abstract:
This article describes two methods for amplifying prions present in experimental and clinical samples: the protein misfolding cyclic amplification (PMCA) assay and the real-time quaking-induced conversion (RT-QuIC) assay. Protocols for preparation of amplification substrate and analysis of results are included in addition to those for the individual assays. For each assay, control and suspect samples are mixed with appropriate amplification substrate, which is whole brains from mice in the case of PMCA and recombinant prion protein produced in bacteria for RT-QuIC, followed by cyclic amplification over a number of cycles of sonication (PMCA) or shaking (RT-QuIC) at a consistent incubation temperature. The resultant amplification products are then assessed either by western blotting (PMCA) or based on fluorescent emissions (RT-QuIC). The equipment and expertise necessary for successfully performing either assay vary and will be important factors for individual laboratories to consider when identifying which assay is more appropriate for their experimental design. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Prion amplification via protein misfolding cyclic amplification Support Protocol 1: Collection of whole brains from mice and preparation of normal brain homogenate Basic Protocol 2: Prion amplification via real-time quaking-induced conversion Support Protocol 2: Preparation of recombinant truncated white-tailed-deer prion protein.
Insights
This study details two prion amplification methods: protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC). Both assays amplify prions from samples, with varying equipment needs for laboratory selection.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Prions are misfolded proteins implicated in neurodegenerative diseases.
- Sensitive detection of prions is crucial for diagnostics and research.
- Existing amplification techniques require optimization for clinical and experimental samples.
Purpose of the Study:
- To describe and compare two prion amplification methods: PMCA and RT-QuIC.
- To provide detailed protocols for assay preparation, amplification, and analysis.
- To guide laboratories in selecting the most appropriate assay based on their resources and experimental needs.
Main Methods:
- Protein Misfolding Cyclic Amplification (PMCA): Uses mouse brain homogenate as substrate, amplification via sonication cycles.
- Real-Time Quaking-Induced Conversion (RT-QuIC): Employs bacterially produced recombinant prion protein as substrate, amplification via shaking cycles.
- Both methods involve incubating samples with substrate, followed by cyclic amplification and analysis via Western blotting (PMCA) or fluorescence (RT-QuIC).
Main Results:
- Successful amplification of prions using both PMCA and RT-QuIC assays.
- Detailed protocols provided for substrate preparation and result analysis for each method.
- Differences in equipment and expertise requirements highlighted for assay selection.
Conclusions:
- PMCA and RT-QuIC are effective methods for amplifying prions in various samples.
- The choice between PMCA and RT-QuIC depends on laboratory-specific factors like available equipment and technical expertise.
- Standardized protocols facilitate reproducible prion detection and research.
