Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT

Circular RNAs (circRNAs) are covalently closed RNA molecules known for their increased stability compared to linear RNAs. Synthetic circRNAs are being developed as RNA therapeutics, while natural circRNAs are being investigated for their biological roles in eukaryotes and their potential as disease biomarkers. Consequently, the accurate detection and validation of circRNAs is crucial for advancements in both fundamental RNA research and biotechnological applications. Common methods for circRNA validation involve RT-PCR using divergent primers, followed by sequencing across the circRNA junction. However, most described methods are high-throughput approaches that require time-consuming RNA processing steps, and they are unable to detect highly structured circRNAs. Additionally, methods for low-throughput sequencing of small circRNAs (<150 nt) require cloning prior to sequencing. A simplified protocol for the validation of circRNA sequences irrespective of structure, sequence complexity, and length has not yet been described. In this method, we describe an improved RT-PCR protocol for circRNA detection by using UltraMarathonRT^® (uMRT), a highly processive reverse transcriptase. Unlike other reverse transcriptases, uMRT can reverse-transcribe large, structured circRNAs of varying sizes, at ambient temperatures, enabling sequencing of the resulting concatemeric amplicons generated by RT-PCR and other methods. Using this method, we sequenced circRNAs containing highly structured internal ribosome entry sites commonly utilized in synthetic circRNAs, natural circRNAs containing repetitive elements, and small circRNAs, all without the need for cloning. With this new platform, we offer a protocol for the precise detection of nearly any circRNA species. Key features • This protocol shortens current methods for circRNA detection and sequencing by sequencing RT-PCR products directly, without the need for cloning or processing the PCR product. • This protocol describes a simple and cost-effective RT-PCR method for single circRNAs. • The highly processive UltraMarathonRT (uMRT) functions at ambient conditions, reducing circRNA degradation. • This protocol enables sequencing of circRNAs that are below 150 nt as well as larger circRNAs. • This protocol enables the detection of structured and very large circRNAs.