Circular RT-PCR Assay Using Arabidopsis Samples

Abstract

Post-transcriptional processing is critical for RNA biogenesis, in which conventional functional RNA transcripts are generated, such as messenger RNAs (mRNAs), transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) for translation as well as emerging non-coding RNAs with known or unknown regulatory functions. To determine the precise termini of an RNA molecule during or after processing, the primer extension and Rapid Amplification of cDNA Ends (RACE) methods have been routinely utilized for the precise mapping of 5’ or 3’ ends. Different from these assays, which are designed to detect only one end of a specific target RNA at a time, circular Reverse Transcription-Polymerase Chain Reaction (cRT-PCR) is able to simultaneously determine both the 5’ and 3’ ends of the target RNA. In Arabidopsis thaliana, cRT-PCR has been wildly applied to identify both the 5’ and 3’ extremities of the ribosomal RNA precursors, or to assess the length or post-transcriptional extensions at the 3’ end of a matured mRNA. In this protocol, we summarize and present a detailed procedure of the cRT-PCR assay in Arabidopsis thaliana, which is also successfully used in our previously published work (Hang et al., 2014).