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

ABSTRACT: Hairpin-containing pre-miRNAs are precursors of microRNAs (miRNAs) that play important roles in cellular processes and various human diseases. Pre-miRNAs are produced from longer primary transcripts (pri-miRNAs). The falsity in cellular expression and sequences of pre-miRNAs might cause cellular defects or human diseases. However, the current pre-miRNA quantification methods by qPCR cannot discriminate between pre-miRNAs and their parental pri-miRNAs. In addition, the ligation of the sequencing adapter to 5’-end of pre-miRNAs is inefficient, therefore, pre-miRNA sequencing is highly impractical. Here, we developed a method, called the intramolecular ligation method (iLIME) for pre-miRNA quantification and sequencing. This method utilized T4 RNA ligase 1 to convert hairpin-pre-miRNAs into circularized RNAs that do not naturally exist in cells. The resulting circuliarized RNAs allow us to design unique primers to quantify pre-miRNAs by qPCR specicially, and thus this qPCR can distinguish pre-miRNA from pri-miRNAs. In addition, the iLIME also allows us to sequence pre-miRNAs using next-generation sequencing. The iLIME method offers a simple and effective way to quantify and sequence pre-miRNAs. This will be useful in investigating pre-miRNAs for addressing research questions for medical applications. The iLIME can be potentially extended to other hairpin-containing RNAs, such as tRNAs and snRNAs.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE185524 | GEO | 2021/10/11

REPOSITORIES: GEO

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

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