Identification of coding and non-coding RNA classes expressed in swine whole blood

Authors: FLEMING, DAMARIUS - Orise Fellow; Anderson, Sarah; Miller, Laura

Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/13/2018
Publication Date: 11/28/2018
Citation: Fleming, D.S., Anderson, S.J., Miller, L.C. 2018. Identification of coding and non-coding RNA classes expressed in swine whole blood. Journal of Visualized Experiments. (141):e58689. https://doi.org/10.3791/58689.
DOI: https://doi.org/10.3791/58689

Interpretive Summary: Gene expression describes the regulation of metabolic processes which involves the expression of a partial copy of the gene's DNA sequence - called messenger RNA or mRNA. The mRNA is used as a template to direct the synthesis of a protein which in turn regulates cellular function. Gene expression profiling is the process of determining which genes are active in a specific cell or group of cells. It is accomplished by measuring mRNA, the intermediary between genes and proteins. A non-coding RNA (ncRNA) is an RNA molecule that is not translated into a protein. Noncoding RNAs belong to several groups and have regulatory roles in cellular processes, they may therefore be attractive new targets for therapeutics. This manuscript is video-article to demonstrate processing of whole blood samples for profiling coding and non-coding RNAs for other researchers to follow.

Technical Abstract: Gene expression describes the regulation of metabolic processes which involves the expression of a partial copy of the gene's DNA sequence - called messenger RNA or mRNA. The mRNA is used as a template to direct the synthesis of a protein which in turn regulates cellular function. Gene expression profiling is the process of determining which genes are active in a specific cell or group of cells. It is accomplished by measuring mRNA, the intermediary between genes and proteins. A non-coding RNA (ncRNA) is an RNA molecule that is not translated into a protein. Noncoding RNAs belong to several groups and have regulatory roles in cellular processes. Abundant and functionally important types of non-coding RNAs include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), as well as small RNAs such as microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs and the long ncRNAs such as Xist and HOTAIR. Many of the newly identified ncRNAs have not been validated for their function. Because these small ncRNA are so important to fundamental biologic processes and contribute significantly to certain pathophysiologic states, they may therefore be attractive new targets for therapeutics. This manuscript demonstrates a modified protocol for the generation coding and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitting size selection, to allow capture of more RNA species.