Detection and Identification of Uncapped RNA by Ligation-Mediated Reverse Transcription Polymerase Chain Reaction
The 5′-cap structure is an essential feature in eukaryotic mRNA required for mRNA stability and enhancement of translation. Ceratin transcripts are selectively silenced by decapping in the cytoplasm and later become translationally active again by acquiring the cap structure to regenerate translatable mRNAs. Identification of uncapped mRNA transcripts will reveal how gene expression is regulated by the mRNA recapping pathway. What follows is a sensitive method to detect and identify the uncapped mRNA from the cells. The technique consists of three parts: selective ligation of anchor RNA to the 5′-end of monophosphate RNA by double-strand RNA ligase, conversion of ligated RNA product into cDNA by reverse transcription, and amplification of a specific cDNA by polymerase chain reaction.
Key wordsRT-PCR 5′-cap Uncapped mRNA mRNA recapping RNA ligation RNA processing Trypanosoma brucei
I thank Bryan Mathis (University of Tsukuba) for scientific editing service. This work was supported by the National Science Foundation under Grant Number 1050984 and the JSPS Grants-in-Aid for Scientific Research KAKENHI 16H05180.
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