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Introduction to Exosomal RNA Sequencing

Exosomes are cell-derived vesicles that can be found in a variety of eukaryotic fluids, including blood, urine, and cell culture medium. Exosomes travel throughout the body after being released, fusing with other cells and transferring their cargo to the acceptor cell. Exosomes appear to play an important role in intercellular communication and disease transmission, according to new research.

Figure 1. The biogenesis of exosomes and the RNAs in exosomes.

Exosomes contain microRNAs, mRNAs, lncRNAs, and other ncRNAs in different subsets. Exosomal delivery of miRNA can result in fully functional or translatable long RNA within a recipient cell, according to growing evidence. Exosomal miRNAs could play a role in cell-cell communication and could be used as biomarkers to detect and monitor disease. Exosomal RNA profiling elucidates the function of exosome-associated RNA and allows for the applications listed below.

• Changes in exosomal miRNA during disease states are thought to be a reliable method for discovering sequence signatures for disease diagnosis and prognosis.
• Exosomal mRNA's identity and expression level have been considered an important approach for identifying biomarkers in blood, saliva, and urine biofluid samples.
• Exosomes contain long noncoding RNAs (lncRNA), which can have expression patterns that differ from those found in cells.

Exosomal RNA Sequencing Categories

Exosomal Small RNA-Seq: Exosomal small RNA sequencing is a powerful tool for analyzing small RNAs, such as miRNAs, piRNAs, and siRNAs, and it provides both quantitative and qualitative data.

Exosomal microRNA Sequencing: Examines monitor global microRNA expression at a low cost, allowing biomarkers linked to diseases like cancer to be identified.

Exosomal Long RNA-Seq: Exosomal long RNA-seq examines the sample's mRNAs, long non-coding RNAs, and circular RNAs, allowing for alternative splicing analysis, the detection of novel transcripts, and gene fusion events.

Exosomal lncRNA-Seq: Exosomal lncRNA sequencing looks at exosome-wide lncRNAs, which can detect promising cancer biomarkers because exosomal lncRNAs are involved in tumorigenesis, tumor angiogenesis, and chemoresistance.

Exosomal mRNA-Seq: With or without prior knowledge of exosomal mRNA sequences, an exosomal mRNA sequencing can not only in profiling exosomal mRNA in terms of expression levels and dynamics but also in comprehending the physiological roles.

Exosomal circRNA-Seq: Exosomal cicrRNA-Seq can obtain global information on exosomal circRNAs quickly and efficiently. circRNAs can be detected from very small amounts of cellular material using our single-base resolution technology.

Exosomal RNA Sequencing Workflow Protocol and Bioinformatics Pipeline

There are four major steps in the exosomal RNA sequencing workflow: (1) sample preparation, which will include RNA purification, quality assessment, and quantification, (2) library preparation, which will include RNA fragmentation, (3) sequencing, and (4) data analysis, which will include visualization and preprocessing of results as well as custom bioinformatics analysis.

The protocol for all RNA-seq workflows is the same. Prior to preparing an RNA library, total RNA is extracted from a biological sample of interest, which may then be purified to enrich mRNAs, microRNAs, and lincRNAs. In single-end sequencing reactions, one read is displayed, while in paired-end reactions, two ends are separated by an unsequenced fragment. In a nutshell, RNA-Seq has given researchers an unprecedented view of the transcriptome in both normal and pathological conditions. For transcriptional profiling assays, NGS is quickly becoming the method of choice. High-throughput sequencing, as opposed to microarray technology, allows for the identification of novel transcripts while avoiding the need for a sequenced genome and avoiding background noise associated with fluorescence quantification.

Exosomal RNA sequencing bioinformatic pipeline includes in-depth analysis of the following: (1) exosomal RNA length distribution and quantitative analysis, (2) differential exosomal RNA expression analysis, (3) exosomal RNA and binding site prediction, (4) GO annotation analysis, (5) KEGG pathway analysis, (6) interactive network analysis, (7) coding-non-coding gene co-expression network (CNC network), and (8) lncRNA-miRNA-mRNA network.

References:

1. Srinivasan S, Yeri A, Cheah PS, et al. Small RNA sequencing across diverse biofluids identifies optimal methods for exRNA isolation. Cell. 2019 Apr 4;177(2).
2. Nassirpour R, Mathur S, Gosink MM, et al. Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms. BMC genomics. 2014 Dec;15(1).
3. t Hoen PA, Friedlander MR, Almlof J, et al. Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories. Nat Biotechnol. 2013;(11).