ligation independent cloning serial cloner

Here, we present a compilation of extant small RNA cloning methods, options for sequencing, and some of the small RNA results that we have obtained in the “still unsequenced” allotetraploid cotton genome. Thus, reliable and reproducible methods for cloning small RNA species are of paramount importance and will remain so into the foreseeable future. On the other hand, de novo finding of miRNAs in species whose genomes have yet to be sequenced and discovering new classes of small RNAs must still rely upon in vitro cloning from purified cellular RNAs. That stable hairpin makes miRNA prediction in sequenced genomes a relatively tractable exercise. Of these small RNA classes, only miRNAs form a characteristic thermodynamically stable hairpin structure. In addition, the universe of small RNAs has expanded to include not only miRNAs but new classes including endogenous small interfering RNAs (siRNAs), 21U RNAs, and Piwi-interacting RNAs (piRNAs). Hence, the discovery of miRNAs represents a hallmark in RNA science for understanding RNA-dependent regulation of many complex biological processes such as development, function of metabolic pathways, cell fate and death. Similarly, the world of miRNAs has grown from the two original nematode “genes” to now number more than one thousand loci in plants and animals and their role in regulating cellular processes has expanded to a point where virtually all normal and pathogenic cellular processes are affected at some point by one or more of these tiny entities. Moreover, RNAi is proving to be a potentially powerful tool in attacking pathogenic cellular processes. Over the past several years RNAi has become a powerful tool for understanding the role played by dozens of plant and animal genes in a wide range of cellular processes, both normal and pathogenic. The two worlds of RNAi and miRNAs merged when it was observed that both RNAi and miRNAs employed the same mechanism to carry out their mission of regulating eukaryotic gene expression. Like RNAi, this phenomenon, initially called short temporary RNA (stRNA), was at first regarded as a one-off curiosity but, again like RNAi, persistence paid off with the explosive validation of the microRNA (miRNA). At the same time, another curious phenomenon was being observed by Victor Ambros, Gary Ruvkun, and colleagues in nematodes. The phenomenon of RNA interference (RNAi) was being uncovered as cosuppression in plants, quelling in fungi, and RNAi in nematodes through the 1990s and at least the broad strokes of the mechanism were elucidated by the turn of the 21st Century. In the 1990s two independent discoveries opened up the previously unsuspected world of noncoding RNAs (ncRNAs). 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. Thus, reproducible methods for in vitro small RNA cloning are of paramount importance and will remain so into the foreseeable future. Nevertheless, for the many “yet-unsequenced” plant genomes, the discovery of small RNA world requires in vitro cloning from purified cellular RNAs. 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. 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. 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.

Ligation independent cloning serial cloner