TY - CHAP
T1 - Analysis of Annotated and Unannotated Long Noncoding RNAs from Exosome Subtypes Using Next-Generation RNA Sequencing
AU - Suwakulsiri, Wittaya
AU - Chen, Maoshan
AU - Greening, David W.
AU - Xu, Rong
AU - Simpson, Richard J.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - Bioinformatics
KW - Exosomes
KW - Long noncoding RNAs
KW - Next-generation RNA sequencing
KW - Transcriptomics
UR - http://www.scopus.com/inward/record.url?scp=85097977865&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-1158-6_12
DO - 10.1007/978-1-0716-1158-6_12
M3 - Chapter (Book)
C2 - 33326077
AN - SCOPUS:85097977865
SN - 9781071611579
T3 - Methods in Molecular Biology
SP - 195
EP - 218
BT - Functional Analysis of Long Non-Coding RNAs
A2 - Cao, Haiming
PB - Humana Press
CY - United States
ER -