Exosomes are small extracellular vesicles that contain RNA, proteins, and lipids. These vesicles play a crucial role in intercellular communication and have been implicated in various physiological and pathological processes. Exosome RNA-Seq is a powerful technique that can be used to study the RNA content of exosomes and gain insight into their function and regulation. In this guide, we will provide an overview of the key steps involved in exosome RNA-Seq and highlight some important considerations for beginners.
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Exosomes were first described in 1981 as nano-vesicles secreted in vitro by different cell types with a lipid composition different from that of the plasma membrane, suggesting a different and more complex origin than simple membrane outgrowth. Exosomes can be secreted by almost all cell types, including immune cells, blood cells, neuronal cells, epithelial cells, and cancer cells. They contain proteins that are retained from primary cells through biogenesis, and in addition, exosomes can contain miRNAs, mRNAs, and even long non-coding RNAs (lncRNAs) that are significantly involved in exosome regulation.
The biogenesis process of exosomes is very complex and can be generally divided into two types: endotrophic exosomes and vesicular exosomes. The precursor material of endotrophic exosomes is synthesized into small vesicles on the endoplasmic reticulum, which then gradually mature into secretable endotrophic exosomes under the mediation of Golgi apparatus and polycystic bodies. In contrast, the biosynthesis of vesicular exosomes takes place at the cell membrane. In this process, the cell membrane forms two different types of vesicular exosomes by inward and outward flipping.
Mechanisms of exosome biogenesis and secretion. (Jadli et al., 2020)
Exosomes contain several types of RNA molecules, such as mRNA, miRNA, lncRNA and circRNA. These RNA molecules are derived from the cytoplasm and nucleus, and they are included in exosomes through different pathways.
In general, RNA molecules can be included in exosomes through several pathways:
The biologically active substances of exosomes can affect the metabolism of receptor cells in two main ways. The first is the direct interaction of exosomal surface proteins with target cell receptors. The second is the internalization of the contents, after fusion with the plasma membrane of the target cell, or through endocytosis and phagocytosis by macrophages. Exosomes are of clinical importance, especially in tumors.
The main role of exosomes is to transmit information between cells of the organism. They participate more generally in the homeostasis of the internal environment of cells and tissues by regulating cell viability, status and function, and they can significantly mediate tissue repair. Exosomes also contribute to tumorigenesis and development, including the transformation of normal cells into malignant cells and angiogenesis.
Principle | Features | |
---|---|---|
PCR | Multi-cycle amplification of DNA fragments using DNA polymerase Detects the presence and abundance of target RNA molecules | Low throughput; Cannot distinguish between different types of RNA |
Microarray | Determining the presence and abundance of RNA molecules by detecting the hybridization signal between RNA and the probe | Detects a large number of RNA molecules; Can only detect known RNAs; Low sensitivity at low expression levels |
RNA-Seq | High-throughput sequencing technology | Transcriptome-wide analysis of RNA, while distinguishing between different types of RNA; Can detect the presence and abundance of all RNA molecules, including unknown RNAs; High throughput, high sensitivity and high resolution; Requires high sequencing depth and computational power |
High-throughput sequencing technology has now become one of the main tools for studying exosomal RNA. Through RNA sequencing technology, the type, quantity and differential expression of exosomal RNAs can be comprehensively and efficiently analyzed to gain insight into the function and mechanism of action of exosomal RNAs in the development of different diseases.
Please read our article Exosomal RNA Sequencing: Introduction, Categories, and Workflow to get more information.
Exosomal RNA research (miRNA/mRNA/circRNA /lncRNA) involves many technical difficulties, including sample collection, RNA purification, library preparation, sequencing techniques and data analysis.
Exosome isolation techniques, contents, and applications. (Li et al., 2019)
The following are some common technical difficulties and points to consider:
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