Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)

The “RNA revolution” that started at the end of the 20th century with the discovery of post-transcriptional gene silencing and its mechanism via RNA interference (RNAi) placed tiny 21-24 nucleotide long noncoding RNAs (ncRNAs) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. The discovery of new classes of ncRNAs including endogenous small interfering RNAs, microRNAs, and PIWI-interacting RNAs is a hallmark in the understanding of RNA-dependent gene regulation. New generation high-throughput sequencing technologies further accelerated the studies of this “tiny world” and provided their global characterization and validation in many biological systems with sequenced genomes. Nevertheless, for the many “yet-unsequenced” plant genomes, the discovery of small RNA world requires in vitro cloning from purified cellular RNAs. Thus, reproducible methods for in vitro small RNA cloning are of paramount importance and will remain so into the foreseeable future. In this paper, we present a description of existing small RNA cloning methods as well as next-generation sequencing methods that have accelerated this research along with a description of the application of one in vitro cloning method in an initial small RNA survey in the “still unsequenced” allotetraploid cotton genome.