Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT
Year: 2025
Type: article
Abstract: (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.
Source: BIO-PROTOCOL
Authors: Ruben Warkentin, Anna Pyle
Institutions: Yale University, Howard Hughes Medical Institute
Language: English
Topic: Circular RNAs in diseases
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W4414524533