Long-term objectives: Modified ribonucleic acids (RNA) are important regulators of numerous cellular processes, and are increasingly being associated with human diseases. For example, non-coding microRNAs play significant roles in neuronal development, and can serve as biomarkers for psychiatric disorders. Another such modified RNA is circular RNA (circRNA), a non-coding transcript present in the cytoplasm. Since circRNAs are present in lower abundance than other RNA molecules and share sequence homology with mRNA, they are difficult to isolate from total RNA. Our long-term objective is to further develop and distribute a molecular biology kit, which will foster broader research in this nascent field and expand our understanding of the role of circular RNAs in neuronal development, substance abuse, and psychiatric disorders. We have established four Phase II Aims: (1) Method optimization, (2) Clinical Sample Applicability + Optimization, (3) Internal Kit Testing/Cofactor circRNA Service, and (4) External Field Testing/ Kit Beta Testers. Research Design:
Aim 1 : We will utilize universal human brain reference RNA containing a known quantity of circRNA control, and our optimized enzyme-mix to determine ideal reaction parameters (i.e. primer design, temperature, and incubation time) that will lead to the most robust enrichment of ciRNA species. Input titrations will determine the threshold and range of total RNA input for the optimized protocol. Success will be measured by qPCR and sequencing metrics on libraries passing our quality control measures.
Aim 2 : We will obtain commercially available biobank samples such as FFPE tissue, frozen brain, and plasma to test the kit applicability and robustness on clinically relevant samples. Quality metrics assayed by Qubit, Bioanalyzer/Tapestation, and Nanodrop will be recorded for the corresponding sequencing libraries to identify kit robustness. In addition, samples provided by commercial and academic partners will further bolster our metric reports for varying sample types and input amounts.
Aim 3 : Our production scientists will translate the R&D SOP into a production service offering with client-submitted samples. We will continue to collect quality metrics on these samples in the production setting to determine expected yields for library construction and circRNA control enrichment. We will use the data generated from both our R&D and production setting to develop and optimize our analysis software, ActiveSite, to incorporate circRNA enrichment reports.
Aim 4 : We have interested commercial and academic partners that have volunteered to test our circRNA kit in their respective laboratories. Our partners will collect quality metric scoes about the samples, generate circRNA- enriched samples, and provide these to us for evaluation and sequencing. We will utilize feedback from these Beta testers to refine the SOP and our market strategy.
Circular RNAs are a recently discovered non-canonical form of RNA that have been shown to interact with microRNAs, molecules which are important in multiple neurological disorders. We are developing a user- friendly kit that will enable a broad range of biomedical researchers to access and study these important molecules in order to further our understanding of their role in biology and human health.
