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).
Keywords: Circular RT-PCR, Post-transcriptional processing, Pre-rRNA processing, Ribosome biogenesis, PRMT3
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from the previously published studies (Barkan, 2011; Slomovic et al., 2008) and Dr. Alice Barkan’s lab homepage (http://pml.uoregon.edu/protocols.html), and it was performed in (Hang et al., 2014). This work was supported by National Natural Science Foundation of China Grants 31330020 and 31210103901 (to X.C.), 31370770 and 31171184 (to C.L.), and 31200900 (to X.D.), and State Key Laboratory of Plant Genomics Grant SKLPG2011B0101.
References
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