Exosome RNA Sequencing

Exosomes are endosome-derived small membrane vesicles with diameters less than 150 nm that are present in nearly all biological fluids (e.g., blood, breast milk, saliva and urine). The exosomes contain not only protein components, but also some RNA components, such as microRNA (miRNA), long non-coding RNA (lncRNA) and messenger RNA (mRNA), and circular RNA (circRNA). These RNAs carried by exosomes are collectively called exosomes-derived RNAs, which have a complete sequence structure and biological activity.

Fig1. Schematic representation of exosome biogenesis and molecular cargo. (Hofmann, 2020)

Overview of Our Exosome RNA-Seq Service

CD Genomics adopts the method of ribosome removed library construction to the whole transcriptome sequencing service of exosomes. We can simultaneously detect lncRNA, mRNA and circRNA in samples to obtain comprehensive transcriptome information, and then carry out expression analysis, structural research, variation research and discovery of new transcripts for different types of RNA.

Project Workflow

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Differential analysis
• Function enrichment analysis
• Prediction and identification of circRNA
• Principal component analysis of samples based on gene expression
• Construction of lncRNA-mRNA jointly express network (CNC network)
• Transcriptional regulatory activity analysis and pathway interaction analysis

Sample Requirements

Sample Volume: For cell supernatant, a minimum quantity of ≥ 30 ml is recommended, while serum and plasma samples should be ≥ 4 ml. Bile samples should be ≥ 5 ml, and urine samples should be ≥ 20 ml. For cerebrospinal fluid, chest fluid, and ascites, a minimum of ≥ 30 ml is advised. Exosomal RNA quantity is ≥ 20 ng for accurate and reliable results.

Please refer to our Exosome RNA Sequencing Sample Submission Guidelines for more details.

Sample Quality: no obvious degradation of RNA, ≥ 500 ng/μl, 1.8 ≤ OD260/280 ≤ 2.2, RIN ≥ 7.0, 28S:18S ≥ 1.5.

Deliverable: FastQ, BAM, QC report, differential expression RNA screening, cluster analysis, target gene enrichment analysis, KEGG/ GO analysis, custom bioinformatics analysis.

Demo Results

Case Studies

Parkinson's disease is a challenging puzzle without effective treatments. In our study, we investigated the molecular issues behind dopaminergic dysfunction using MSC exosomes. Whole transcriptome sequencing on MSC exosomes, with a focus on miRNA profiles, was employed. Subsequent analysis of genes through GO and KEGG pathway studies provided valuable insights. The analysis pointed to the involvement of key pathways, specifically PI3K-Akt and AMPK, known to be associated with Parkinson's disease and neurodegeneration. A potential target, Nox4, was identified, exploring how miR-100-5p could regulate it. This interaction was confirmed through qPCR and dual luciferase assays. This discovery enhances understanding and suggests potential targets for future therapies.

Sequencing analysis of T-MSCs-Exo miRNAs, and miR-100-5p directly targets the 3' UTR NOX4. (He et al., 2023)

FAQ

• • What kinds of method should we use for building a whole transcriptome library from 100-200ng of exosomal RNA?

    • If you have more than 100 ng of RNA, it's recommended to use the conventional method for optimal gene information coverage. We provide library which is better suited for small samples, so choose the conventional method whenever possible for larger experiments.

      Please refer to our Exosome RNA Sequencing Sample Guidelines for more information about Exosome RNA-Seq sample submission.

• • When conducting exosome studies, should plasma or serum be utilized?

    • Plasma and serum serve distinct roles in exosome studies. Plasma is the liquid component of blood devoid of cells, while serum is plasma minus fibrinogen and clotting factors. The choice depends on the experiment's nature. Generally, plasma is preferred for standard experiments due to its composition. However, in specific cases, such as those involving platelet-related diseases, serum might be more suitable, as it contains fewer clotting components and exhibits unique exosome characteristics resulting from platelet secretion during clotting.

      You can refer to our Sample Submission and Preparation Guidelines for more information.

• • How can serum-derived exosomes be eliminated when culturing cells?

    • When utilizing serum in in vitro cell culture, it often comes with the presence of exosomes, risking contamination of cell-derived exosomes. To address this concern, two effective methods can be employed:

      (1) Ultracentrifugation: Serum designated for cell culture can undergo ultracentrifugation at 100,000 g for 10 hours, effectively removing serum-derived exosomes.

      (2) Serum-Free Medium: Opt for serum-free medium in cell culture to completely eliminate the presence of serum-derived exosomes.

      These approaches help maintain the purity of cell-derived exosomes for more accurate and reliable experimental results.

• • Does the presence of dead cells impact exosome extraction during cell culture?

    • Yes, it does. When collecting cells, it's crucial to ensure that the percentage of dead cells remains below 5%. The apoptotic or dead cell process releases a significant number of vesicles of diverse sizes. These can potentially introduce contamination into the extracted exosomes, produced by the viable cells, during the exosome extraction and purification process. Therefore, maintaining a low percentage of dead cells is essential for obtaining high-quality and uncontaminated exosome samples.

• • What is the recommended initial sample volume for preparing exosome small RNA sequencing?

    • For common tumor cell lines, it's advised to start with an initial sample volume of 40 mL or more. However, in cases where the exosome content is low, such as in suspension cells, stem cells, or neuronal cells, a preliminary concentration step using a 10kD ultrafiltration column is recommended. After concentrating to 40 mL or more, proceed with ultracentrifugation to separate the exosomes. In general, it is necessary to extract more than 20 ng of Total RNA for optimal results.

• • Which internal reference is recommended for conducting exosomal RNA RT-PCR experiments?

    • The choice of internal reference depends on the specific sample characteristics. For optimal results, consider the following options:

      1. Exogenous Addition: Introduce cel-miR-39-3p externally.

      2. Endogenous Options: Choose from endogenous references like U6, SNORD61, SNORD68, or SNORD72 based on the specific requirements of your experiment.

      Tailoring the internal reference to your sample type ensures accurate and reliable results in exosomal RNA RT-PCR experiments.

• • How can extracted exosomes be identified?

    • Extracted exosomes are typically identified through multiple methods, ensuring a comprehensive analysis:

      1. Transmission Electron Microscopy (TEM): Utilized for morphology detection, offering visual insights into exosome structure.

      2. Particle Size Detection: Determines size distribution, providing crucial information about exosome dimensions.

      3. Western Blot Detection: This involves the identification of exosome marker proteins, including CD63, CD9, CD81, TSG101, etc. Following the International Society for Extracellular Vesicles (ISEV) guidelines, the detection of two positive markers and one negative indicator in Western blot assays is recommended for robust confirmation.

      By combining these techniques, a thorough and reliable assessment of extracted exosomes can be achieved.

References:

1. Hofmann, Linda, et al. The Emerging Role of Exosomes in Diagnosis, Prognosis, and Therapy in Head and Neck Cancer. International Journal of Molecular Sciences 21.11(2020):4072.
2. Li, Yan, et al. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis. Nature Publishing Group 8(2015).
3. He, Songzhe et al. "miR-100a-5p-enriched exosomes derived from mesenchymal stem cells enhance the anti-oxidant effect in a Parkinson's disease model via regulation of Nox4/ROS/Nrf2 signaling." Journal of translational medicine vol. 21,1 747. 24 Oct. 2023.

Exosomal Small RNA Sequencing

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Exosomal lncRNA Sequencing

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Exosomal microRNA Sequencing

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Exosomal circRNA Sequencing

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Exosomal Long RNA Sequencing

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Exosomal mRNA Sequencing

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