Modified ribonucleic acids (RNA) are important regulators of translation and other cellular pathways. Non- coding RNAs like 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. To facilitate identification and examination of these molecules, we propose to develop a streamlined approach that uses selective reduction of linear and ribosomal RNAs combined with a novel reverse transcriptase to enrich for circular RNAs. To optimize for robust reduction and amplification, we will test a range of conditions and assess their relative efficiencies using qRT-PCR specific for known circRNAs and synthetic spiked-in linear RNAs. We will use our validated approach to generate circRNA profiles of multiple brain tissues, which will be of significant benefit to researchers interested in understanding the role of circRNA in neuronal development, substance abuse, and psychiatric disorders. As there is no kit on the market which addresses the need for easy circRNA enrichment, development of this protocol will foster broader research in this nascent field and further understanding of the role of circRNAs.
We propose to develop the first commercially available kit for easy isolation and study of circular RNAs. Circular RNAs are recently discovered non-traditional RNAs that interact with microRNAs, molecules which are important in multiple neurological disorders. Developing this kit will enable a broad range of biomedical researchers to study these important molecules and further our understanding of their role in biology and human health.