RNA Captor: A Tool for RNA Characterization

Christian Clepet,Juan Mata

doi:10.1371/journal.pone.0018445

Abstract

BackgroundIn the genome era, characterizing the structure and the function of RNA molecules remains a major challenge. Alternative transcripts and non-protein-coding genes are poorly recognized by the current genome-annotation algorithms and efficient tools are needed to isolate the less-abundant or stable RNAs.ResultsA universal RNA-tagging method using the T4 RNA ligase 2 and special adapters is reported. Based on this system, protocols for RACE PCR and full-length cDNA library construction have been developed. The RNA tagging conditions were thoroughly optimized and compared to previous methods by using a biochemical oligonucleotide tagging assay and RACE PCRs on a range of transcripts. In addition, two large-scale full-length cDNA inventories relying on this method are presented.ConclusionThe RNA Captor is a straightforward and accessible protocol. The sensitivity of this approach was shown to be higher compared to previous methods, and applicable on messenger RNAs, non-protein-coding RNAs, transcription-start sites and microRNA-directed cleavage sites of transcripts. This strategy could also be used to study other classes of RNA and in deep sequencing experiments.

Highlights

The RNA world is appearing more and more complex and deciphering the growing list of RNA species, isoforms and byproducts is a major challenge in biology [1]
Contrary to previous methods [9,10,12], RNA ligase 2 (Rnl2) is used to add an oligoribonucleotide to the 59 end or to the cap site of RNAs
The adapter-ligated RNA can be reverse transcribed with an oligo-dT-primer carrying a second adapter, leading to cDNAs with cloning sites integrated at both ends

Summary

Introduction

The RNA world is appearing more and more complex and deciphering the growing list of RNA species, isoforms and byproducts is a major challenge in biology [1]. Genome annotation algorithms are still limited and full-length cDNAs or RACE PCR remain essential for studying the structure and function of the genes. These approaches are widely used for single gene as well as large-scale genomic programs The main limitation full-length cDNA or 59 RACE methods try to resolve is binding a known sequence at the cap site, so as to prime second-strand polymerization of the cDNA. In the framework of the grapevine genome program, 5 full-length cDNA libraries were constructed with this method and 84,000 clones were sequenced. In the genome era, characterizing the structure and the function of RNA molecules remains a major challenge.

Methods

Results

Conclusion