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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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.
Mechanisms of exosome biogenesis and secretion. (Jadli et al., 2020)
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
|
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.
6 Tips to Make Your Exosome RNA-seq Experiments a Success
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:
- Sample collection: The exosomal RNA content is very low and a large number of samples are required to extract a sufficient amount of RNA. Sample collection, preservation, transfer and handling need to be strictly controlled to avoid RNA degradation and contamination. In contrast, conventional methods to isolate exosomes have low specificity and purity, carry a large number of other impurities, as well as hemeproteins that can encapsulate large amounts of miRNA, severely affecting subsequent RNA and proteomics results. Due to the extremely small amount of exosomal RNA and its specificity, conventional RNA extraction and quality control cannot meet the requirements of subsequent experiments. You can read our article General Considerations and Recommends Exosome Sampling and Pre-processing for more details about Exosome sampling.
- Exosome identification: The International Vesicle Society has strict requirements for exosome identification, emphasizing that exosome identification should include both individual characterization and overall characterization, and that these characterizations are highly demanding in terms of equipment and operation. Currently, the main methods for exosome identification include, 1) observation of the presence of exosome-like structures in the sample by electron microscopy; 2) particle size detection of exosomes, and 3) detection of exosome surface antigen markers by flow cytometry.
- RNA purification: The purification of exosomal RNA is also a key technical difficulty. Exosomal RNA is present in complex samples, such as plasma, urine, saliva and cell culture supernatants, and requires sample pretreatment and enrichment followed by RNA purification to obtain sufficient RNA quantity and quality. Conventional isolation methods cause physical shear damage and introduce chemical contamination, affecting subsequent functional studies.
- Research technique: qPCR of exosomal RNA faces the practical difficulties of low nucleic acid quantity, difficult quality control system, and how to select internal and external reference. High-throughput sequencing technology is usually used for exosomal RNA, but different technologies will have different advantages and disadvantages, and the appropriate technology needs to be selected according to the specific research questions.
- Library preparation: Due to the low content and varying fragment length of exosomal RNA samples, library preparation requires multiple steps of optimization. Conventional library construction methods cannot be adapted to exosomal RNA, resulting in low library quality, low sequencing data comparison rate and biased results; for example, RNA fragment length selection, RNA reverse transcription, RNA amplification, RNA labeling and library purification are required to obtain high quality libraries.
- Data analysis: Exosome RNA sequencing data processing requires a large number of data cleaning, quality control, comparison and annotation steps, as well as the analysis and interpretation of different types of RNAs, which is also a technical difficulty. Some studies may cause depletion of valuable clinical samples due to lack of experience in clinical sample processing.
References:
- Li, Irene, and Barzin Y. Nabet. "Exosomes in the tumor microenvironment as mediators of cancer therapy resistance." Molecular cancer 18 (2019): 1-10.
- Jadli, Anshul S., et al. "Inside (sight) of tiny communicator: exosome biogenesis, secretion, and uptake." Molecular and cellular biochemistry 467 (2020): 77-94.
