Intramolecular ligation method (iLIME) for pre-miRNA quantification and sequencing

• Main Authors: Le, C.T (Author), Le, M.N (Author), Nguyen, T.A (Author)
• Format: Article
• Language: English
• Published: Cold Spring Harbor Laboratory Press 2022
• Subjects: high throughput sequencing; High-Throughput Nucleotide Sequencing; human; Humans; metabolism; microRNA; MicroRNAs; pre-miRNA; pre-miRNA quantification; pre-miRNA sequencing; pri-miRNA; real time polymerase chain reaction; Real-Time Polymerase Chain Reaction
• Online Access: View Fulltext in Publisher
• ISBN: 13558382 (ISSN)
• DOI: 10.1261/rna.079101.122

Hairpin-containing pre-miRNAs, produced from pri-miRNAs, are precursors of miRNAs (microRNAs) that play essential roles in gene expression and various human diseases. Current qPCR-based methods used to quantify pre-miRNAs are not effective to discriminate between pre-miRNAs and their parental pri-miRNAs. Here, we developed the intramolecular ligation method (iLIME) to quantify and sequence pre-miRNAs specifically. This method utilizes T4 RNA ligase 1 to convert pre-miRNAs into circularized RNAs, allowing us to design PCR primers to quantify pre-miRNAs, but not their parental primiRNAs. In addition, the iLIME also enables us to sequence the ends of pre-miRNAs using next-generation sequencing. Therefore, this method offers a simple and effective way to quantify and sequence pre-miRNAs, so it will be highly beneficial for investigating pre-miRNAs when addressing research questions and medical applications. © 2022 Le et al.