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.

What Are Exosomes?

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.

Exosome Biogenesis

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.

Sources of RNAs in Exosomes

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:

• Cytoplasmic RNA: Most of the RNA molecules in exosomes are derived from the cytoplasm of the donor cells. This includes mRNA and some non-coding RNAs that are involved in regulating gene expression.
• Nuclear RNA: Some RNA molecules, such as some long non-coding RNAs, are transcribed in the nucleus and transported to the cytoplasm before being packaged into exosomes.
• RNA degradation products: RNA molecules undergo constant turnover and degradation, leading to the production of small RNA fragments such as miRNAs and piRNAs. These can be packaged into exosomes and transferred between cells.
• Extracellular RNA: Exosomes can also contain RNA molecules from external sources, such as microbial RNA or dietary RNA. These RNA molecules can enter cells and be packaged into exosomes, playing a role in intercellular communication.

Biological Functions of Exosomes

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.

Qualitative and Quantitative Techniques for The Analysis of Exosomal RNA

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.