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Cell lines propagating prions are an efficient and useful means for studying the cellular and molecular mechanisms implicated in prion disease. Utilization of cell-based models has led to the finding that PrP C and PrP Sc are released from cells in association with extracellular vesicles known as exosomes. Exosomes have been shown to act as vehicles for infectivity, transferring infectivity between cell lines and providing a mechanism for prion spread between tissues. Here, we describe the methods for generating a prion-propagating cell line with prion-infected brain homogenate, cell lysate, conditioned media, and exosomes and also detection of protease-resistant PrP with the prion-infected cell assay.

Extracellular vesicles, such as exosomes and microvesicles, have been shown to contain potential microRNA (miRNA) biomarkers that may be utilized in the diagnosis of various diseases from cancer to neurological disorders. The unique nature of the extracellular vesicle bilayer allows miRNA to be protected from degradation making it an ideal source of material for biomarkers discovery from both fresh and archived samples. Here we describe the quantitative analysis of miRNA isolated from exosomes by quantitative PCR and digital PCR.

Prion diseases or transmissible spongiform encephalopathies are disorders of the central nervous system that affect both humans and animals. The underlying cause of prion diseases is the formation and propagation of the infectious prion protein. Prion diseases are difficult to diagnose and treat due to a prolonged asymptomatic incubation period prior to the onset of clinical symptoms. MicroRNAs (miRNAs) are small noncoding RNA species and have been identified as potential biomarkers that also function to regulate disease-specific pathways and proteins in several neurodegenerative disorders, including prion diseases. Here we describe the quantitative analysis of miRNA isolated from neuronal cells infected with a strain of mouse-adapted human prions. These methods can also be adapted to the discovery of miRNA biomarkers in extracellular vesicles, tissue, and noninvasive biological fluids.

Next-generation deep sequencing (NGS) technology represents a powerful and innovative approach to profile small RNA. Currently, there are a number of large-scale and benchtop sequencing platforms available on the market. Although each platform is relatively straightforward to operate, constructing cDNA libraries can be the most difficult part of the NGS workflow. Constructing quality libraries is essential to obtaining a successful sequencing run of high-quality reads and coverage. The quality and yield of RNA affect hybridization and ligation of sequencing adapters. In the field of biomarker discovery, there has been an interest in profiling exosomal RNA from biological fluids. However, very little RNA yield is obtained when extracting RNA from exosomes, thus making library construction difficult. Here, this protocol describes an optimized protocol for constructing small RNA libraries from low yields of RNA, in particular, extracted from exosomes isolated from biological fluids.

In vitro translation systems derived from a wide range of organisms have been described in the literature and are widely used in biomedical research laboratories. Perhaps the most robust and efficient of these cell-free systems is that derived from Escherichia coli. Over the past decade or so, experimental strategies have been developed which have enhanced the efficiency and stability of E. coli cell-free systems such that we can now prepare recombinant proteins on a scale suitable for purification and analysis by biophysical and structural biology techniques, which commonly require relatively large quantities of protein. This chapter describes in detail the protocols employed in our laboratory to prepare translationally active E. coli extracts and to synthesise proteins on a milligram scale from these extracts.

Within the spectrum of sporadic human transmissible spongiform encephalopathies (TSEs), there is considerable diversity of disease phenotypes. At least part of this variation is thought to be on the basis of different "strains" of prions (the infectious agent). Tissue deposition of PrP(res) (the abnormal disease-associated conformation of the prion protein) is considered a hallmark of TSE pathology, and it can be visualized by Western blotting typically as three bands depicting the diglycosylated, monoglycosylated, and unglycosylated species. It is the mobility of the unglycosylated PrP(res), and the relative abundance of the two glycosylated bands, along with the prion protein gene (PRNP) codon 129 genotype, that seem to correlate with distinct clinico-pathological profiles of sporadic Creutzfeldt-Jakob disease.

Prion-propagating cell lines are an efficient and useful means for studying the cellular and molecular mechanisms implicated in prion disease. Use of cell-based models has lead to the finding that prion protein (PrP(C)) and PrP(Sc) are released from cells in association with exosomes. Furthermore, exosomes have been shown to act as vehicles for infectivity, transferring PrP(Sc) between cell lines and providing a mechanism for prion spread between tissues. As a role for exosomes in prion disease is emerging, this chapter outlines a method for the generation of prion-infected cell lines and the isolation and characterization of PrP(C)- and PrP(Sc)-containing exosomes.

The availability of recombinant prion proteins (recPrP) has been exploited as a model system to study PrP-mediated toxicity, conversion, and infectivity. According to the protein only hypothesis, the central event in the pathogenesis of prion diseases is the conversion of PrP(C) to PrP(Sc). This involves a dramatic increase in beta sheet conformation as PrP(C) is converted to PrP(Sc), and it is widely believed that this conformational change affects the as-yet undefined function of PrP(C). Although there are many methods available to monitor for the changes in the structural makeup of PrP mutants and oligomers formed with respect to disease relevance, circular dichroism is one of the most popular methods used. In this chapter, we discuss the fundamental principles of circular dichroism and its current role and applications in prion disease research.