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J Vis Exp. 2018 Nov 28;(141). doi: 10.3791/58689.

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood.

Author information

1
Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service; ORAU/ORISE.
2
Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service.
3
Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service; laura.miller@ars.usda.gov.

Abstract

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA 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 omittedĀ size selection, to allow capture of more RNA species.

PMID:
30582594
DOI:
10.3791/58689
[Indexed for MEDLINE]

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