Efficient circRNA Detection Using the Processive Reverse Transcriptase uMRT.

Area of Science:

• Molecular Biology
• RNA Biology
• Biotechnology

Background:

• Circular RNAs (circRNAs) are stable RNA molecules with roles in gene regulation and disease.
• Accurate detection and validation of circRNAs are critical for RNA research and therapeutic applications.
• Existing circRNA validation methods are often time-consuming, lack throughput, or fail to detect structured or small circRNAs.

Purpose of the Study:

• To develop a simplified, cost-effective, and versatile RT-PCR protocol for circRNA detection and sequencing.
• To overcome limitations of current methods in handling structured, repetitive, or small circRNAs.
• To enable precise sequencing of diverse circRNA species without complex sample processing.

Main Methods:

• Utilized UltraMarathonRT (uMRT), a highly processive reverse transcriptase, for RT-PCR.
• Developed a protocol for direct sequencing of RT-PCR amplicons without cloning or further processing.
• Applied the method to detect and sequence various circRNAs, including structured, repetitive, and small (<150 nt) species.

Main Results:

• The uMRT-based protocol efficiently detected and sequenced circRNAs irrespective of structure, length, or sequence complexity.
• The method enabled sequencing of small circRNAs (<150 nt) and large, structured circRNAs without prior cloning.
• The protocol significantly shortened processing time compared to existing high-throughput methods.

Conclusions:

• This novel RT-PCR protocol offers a precise, efficient, and simplified approach for circRNA detection and sequencing.
• The method is broadly applicable to various circRNA types, facilitating fundamental research and biotechnological applications.
• The protocol's ability to handle diverse circRNA structures and sizes advances the field of RNA therapeutics and biomarker discovery.