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Analysis of annotated and unannotated long noncoding RNAs from exosome subtypes using next-generation RNA sequencing

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posted on 18.03.2021, 03:13 by Wittaya Suwakulsiri, Maoshan Chen, David Greening, Rong Xu, Richard Simpson
Long noncoding RNAs (lncRNAs) contain >200 nucleotides and act as regulatory molecules in transcription and translation processes in both normal and pathological conditions. LncRNAs have been reported to localize in nuclei, cytoplasm, and, more recently, extracellular vesicles such as exosomes. Exosomal lncRNAs have gained much attention as exosomes secreted from one cell type can transfer their cargo (e.g., protein, RNA species, and lipids) to recipient cells and mediate phenotypic changes in the recipient cell. In recent years, many exosomal lncRNAs have been discovered and annotated and are attracting much attention as potential markers for disease diagnosis and prognosis. It is expected that many exosomal lncRNAs are yet to be identified. However, characterization of unannotated exosomal RNAs with non-protein-coding sequences from massive RNA sequencing data is technically challenging. Here, we describe a method for the discovery of annotated and unannotated exosomal lncRNA. This method includes a large-scale isolation and purification strategy for exosome subtypes, using the human colorectal cancer cell line (LIM1863) as a model. The method inputs RNA sequencing clean reads and performs transcript assembly to identify annotated and unannotated exosomal lncRNAs. Cutoffs (length, number of exon, classification code, and human protein-coding probability) are used to identify potentially novel exosomal lncRNAs. Raw read count calculation and differential expression analysis are also introduced for downstream analysis and candidate selection. Exosomal lncRNA candidates are validated using RT-qPCR. This method provides a template for exosomal lncRNA discovery and analysis from next-generation RNA sequencing.

Funding

W.S. is supported by a LaTrobe University Postgraduate Scholarship. R.J.S. and R.X are supported by La Trobe University and D.W.G, in part, by the National Health and Medical Research Council of Australia project grants #1057741 and #1139489.

History

Publication Date

01/01/2021

Book Title

Functional analysis of long non-coding RNAs: methods and protocols

Editors

Cao, H

Publisher

Humana Press

Place of publication

New York

Series

Methods in Molecular Biology

Volume

2254

Pagination

195-218

ISBN-13

9781071611586

Rights Statement

The Author reserves all moral rights over the deposited text and must be credited if any re-use occurs. Documents deposited in OPAL are the Open Access versions of outputs published elsewhere. Changes resulting from the publishing process may therefore not be reflected in this document. The final published version may be obtained via the publisher’s DOI. Please note that additional copyright and access restrictions may apply to the published version.

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