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Exosomes microRNA Sequencing Service

Send us your serum or plasma samples and let our Next Generation Sequencing (NGS) experts perform your NGS analysis in state-of-the-art automated laboratories with rigorous quality control and fast turn-around times and superior data analysis and interpretation. Exiqon offers a complete sequencing service including RNA isolation, library preparation, sequencing, data analysis and biological interpretation of the results in a comprehensive and easy-to-read format.

Exiqon Exosomes microRNA NGS Services

  • Complete sample-to-answer solution covers everything from initial consultation to thorough data analysis and interpretation
  • Provide the sample, and we will take care of exosome isolation, RNA isolation, library preparation, sequencing, data analysis and reporting
  • Close consultation with Exiqon's RNA and NGS experts throughout the process
  • > 10 years' experience with microRNA profiling services
  • Get high quality results from as little as 500 µL serum/plasma
  • Unique and proprietary protocols optimized for exosomes – exosome isolation, RNA isolation, sample QC, library preparation
  • > 50 RNA spike-ins included for QC
  • Fast turn-around times – delivery of the final report within 6-8 weeks
  • Combine with downstream validation by market-leading LNA™-enhanced qPCR platform

A sample-to-answer solution for NGS Service

Our Exosomes microRNA NGS service includes every step in the process: initial consultation with our microRNA and biofluids experts, exosome isolation, RNA isolation, RNA QC, library preparation and QC, sequencing and QC, data analysis and reporting. All analyses are performed by PhD-level scientists who will ensure that you get the best service throughout the project.

A key specifications for the Exosomes microRNA Sequencing Service are shown in Table 1. Libraries are size selected to maximize the number of relevant reads.

Table 1. Key specifications for the Exosomes microRNA Sequencing Service Key specifications for the Exosomes microRNA Sequencing Service

Exiqon Services provide outstanding data analysis, interpretation and visualization. Our superior bioinformatics pipeline is based on solid biological expertise in microRNA and small RNA research.

Data and results from your microRNA profiling project will be delivered in an easy-to-read report with publication grade illustrations including a data analysis files. The complete encrypted raw data can be supplied. Every project concludes with a scientific follow-up discussion with our NGS experts, to answer any questions you may have about your results.

The workflow of an Exiqon Exosomes microRNA sequencing Services project is summarized in Figure 1. Details of each step in the process are described in the following tab.

Exosomes microRNA sequencing

Sequencing microRNA in exosomes is particularly challenging. In performing microRNA-Seq from exosomes, Exiqon takes advantage of years of in-house research experience in working with biofluid samples. Exiqon pioneered microRNA biomarker discovery in serum and plasma and knows what the challenges are and how to overcome them. Over the years, we have performed thousands of RNA extractions and expression profiling experiments from biofluids and exosomes.

Our Exosomes microRNA NGS Service includes unique and proprietary protocols for:
  • High quality isolation of exosomes followed by RNA isolation from serum and plasma samples. Please contact us for more information on sequencing of exosomes from other biofluids such as CSF, saliva and urine
  • LNA™ qPCR-based QC to monitor microRNA content and inhibition
  • Library preparation protocols optimized for biofluids with limited RNA content
  • QC of sequencing data reproducibility and linearity using > 50 RNA spike-ins
microRNA sequencing provides a comprehensive analysis of all microRNAs in the sample. Library size selection (15 – 30 nt) maximizes the number of microRNA reads. Our service includes novel microRNA discovery as well as isomiR counts, enabling you to see precisely which microRNA sequences are expressed in your samples.

Exosomes microRNA sequencing is offered for human serum / plasma samples and other species or other biofluids (e.g. urine, exosomes) upon request.

Figure 2. microRNA Biomarker discovery workflow microRNA Biomarker discovery workflow

Exiqon's Exosomes microRNA profiling services support the discovery and validation of new non-invasive biomarkers for a range of diseases. Biomarker discovery projects usually begin with a genome wide screening phase on a limited number of samples, performed either by a hypothesis-free Next Generation Sequencing (NGS) approach, or by PCR profiling. From this genome wide screening dataset, a smaller number of relevant RNAs are identified, and the discovery phase can continue by analyzing this subset of RNAs on a larger number of individuals, using Focus or custom Pick-&-Mix PCR panels (available from Exiqon as Products or Services). Once a biomarker signature has been identified (including relevant endogenous control RNAs), it is then validated on a large number of individuals using custom Pick-&-Mix PCR panels. Crucially, assays to detect any novel RNAs identified by NGS may also be included in the custom PCR panels.

Contact us for more information
Figure 1 Workflow of an Exosomes microRNA sequencing services project
Workflow of an Exosomes microRNA sequencing services project. (Click to learn more)

1. Consultation

When you engage in a sequencing Service project with Exiqon, you are assured direct communication with the scientists performing your experiments throughout the duration of the project.

Each project begins with a free consultation with a microRNA expression profiling expert. Together we design an experimental setup that best satisfies your research needs and budget. Following this we will complete a detailed sample submission form making sure that all experimental details and subsequent analysis are clearly defined and understood by both parties.

2. Biofluid sample submission

High quality samples are important for accurate microRNA sequencing. At the initial consultation, we offer recommendations on sample collection and handling (especially crucial for biofluid samples) to minimize any pre-analytical variables.

For microRNA-seq in exosomes, the exosome isolation and RNA isolation is included in the project and has to be performed by Exiqon. Exiqon has developed an optimized NGS workflow with proprietary steps including unique RNA isolation and QC protocols specifically optimized for microRNA sequencing from exosomes in serum/plasma.

We recommend sending 500 µl – 1,5 mL of serum or plasma. We accept human serum/plasma sample, and samples from other species or other biofluids (urine, exosomes) upon request.

Please find our recommendations for sample collection in the Biofluids Guidelines.

3. Sample preparation

Exosomes are isolated from biofluids using miRCURY™ RNA Isolation Kits followed by RNA isolation using a proprietary protocol developed by Exiqon specifically for biofluid samples.

4. RNA sample quality control (QC)

QC of the RNA is performed by qPCR QC, using a unique combination of RNA spike-ins and endogenous microRNAs to detect outlier samples and monitor RNases, RNA isolation efficiency, hemolysis, and presence of inhibitors that may affect library preparation. You will receive a report with the RNA-QC results prior to the library preparation and sequencing.

5. Library preparation

Following quality control, we generate libraries and select size using an automated gel cutter allowing customized size selection. The selected size range (15 – 30 nt) maximizes the number of microRNA reads.

6. Sequencing

Exosome microRNA sequencing is performed using Illumina instruments, 1x 50bp reads, 10M reads per sample.

7. Analysis & interpretation

Comprehensive data Analysis including statistical analysis:
  • QC of raw data
  • For Exosomes microRNA sequencing projects, QC includes analysis of >50 spike-ins to check the reproducibility and linearity of the sequencing data
  • Mapping sequences to the appropriate reference genome
  • QC of mapped data
  • Mapping to miRBase and identification of predicted novel microRNAs
  • IsomiR counts can be reported
  • Differential expression analysis to identify known and novel microRNAs that are differentially expressed between your sample groups (unlimited number of group comparisons). Statistical analysis is performed, provided there are a sufficient number of samples per group
  • Unsupervised analysis: Principal Component Analysis (PCA plot) and heatmap
  • Supervised analysis: For each specific group comparison we generate a Volcano plot to display microRNAs which are differentially expressed between the groups, list differentially expressed microRNAs as well as identify statistically significant signals
  • Biological interpretation through use of Exiqon’s knowledgebase (miRSearch) to identify validated and predicted target genes, and microRNAs sharing target genes
  • Gene Ontology (GO) enrichment analysis is performed to identify GO terms that are over-represented within the predicted and validated target genes of the microRNAs that are differentially expressed. This gives a picture of the impact of the experiment on biological pathways and processes
In connection with large service projects we also offer extensive customized bioinformatics analysis.

8. Report and final consultation

The final report is delivered as link to our secure webserver and contains:
  • An easy-to-read data report as pdf containing a description of the project, assessments of sample and data quality and an overview of the results of the data analysis with publication-grade illustrations.
  • Data analysis file which is sufficient for publication, or performing your own analysis of the data if needed.
  • The complete encrypted raw data can be provided on USB3-stick at extra charge.
  • Expected turnaround time: 6-8 weeks
The project concludes with a scientific follow-up discussion to answer any questions you may have about your report and discuss the significance of your results and next steps. Exiqon scientists can also help you design appropriate LNA™ qPCR assays to validate your NGS data (including any novel microRNAs identified), or LNA™ tools to study the function of the candidate microRNAs of interest identified in the study.

Contact us for more information
Figure 1 Workflow of an Exosomes microRNA sequencing services project
Workflow of an Exosomes microRNA sequencing services project. (Click to learn more)

Comprehensive report including biological interpretation

Based on our extensive microRNA and bioinformatics expertise we have developed a unique pipeline for collecting, analyzing and visualizing NGS results.

At the end of each project, you will receive a comprehensive and easy-to-read final report.

Download a report example

The experimental and bioinformatics pipelines are closely integrated, enabling us to provide powerful data analysis and a final report tailored to your specifications. In addition to standard NGS data analysis, the report includes:
  • QC includes analysis of >50 spike-ins which are used to check the reproducibility and linearity of the sequencing data
  • Identification of microRNAs including prediction of novel microRNAs
  • IsomiR counts can be reported
  • Differential expression analysis to identify known and novel microRNAs that are differentially expressed between your sample groups (unlimited number of group comparisons). Statistical analysis is performed, provided there are a sufficient number of samples per group
  • Unsupervised analysis: Principal Component Analysis (PCA plot) and heatmap
  • Supervised analysis: For each specific group comparison we generate a Volcano plot to display microRNAs which are differentially expressed between the groups, list differentially expressed microRNAs as well as identify statistically significant signals
  • Biological interpretation through use of Exiqon’s knowledgebase (miRSearch) to identify validated and predicted target genes, and microRNAs sharing target genes
  • Gene Ontology (GO) enrichment analysis is performed to identify GO terms that are over-represented within the predicted and validated target genes of the microRNAs that are differentially expressed. This gives a picture of the impact of the experiment on biological pathways and processes
In connection with large service projects we also offer fully extensive customized bioinformatics analysis.

After you have received your report, we arrange a scientific follow-up discussion to answer any questions you may have about your report and discuss the significance of your results and next steps. Exiqon scientists can also help you design appropriate LNA™ qPCR assays to validate your NGS data (including any novel microRNAs identified), or LNA™ tools to study the function of the candidate microRNAs of interest identified in the study.

Contact us to discuss your project.

Why study exosomes?

Exosomes are cell derived membranous particles ranging in size from 20 to 120 nm, approximately the same size as viruses but considerably smaller than microvesicles (Figure 1). Exosomes are excreted from cells into the surrounding media and can be found in many if not all body fluids. Their proposed role as intercellular hormone-like messengers together with their stability as carrier of proteins and RNA make them ideal as biomarkers for a variety of diseases and biological processes (Figure 2).

Exosomes are secreted by most cell types and are formed by the fusion of multivesicular bodies with the plasma membrane. They are believed to be involved in a number of functions, including:
  • Immune regulation (e.g. tumor derived exosomes may help the tumor to evade the immune response)
  • Blood coagulation
  • Cell migration
  • Cell differentiation
  • Cell-to-cell communication
Microvesicles that are larger than exosomes (up to 1 µm) are typically formed by blebbing of the plasma membrane, whereas exosomes are released by exocytosis from multivesicular bodies of the endosome.

How to isolate exosomes

The two classical ways to isolate exosomes are either by cumbersome sucrose gradient ultra-centrifugation or by isolation using antibodies specific for one of the surface proteins typically found on exosomes. The last method is very specific and might exclude some sub fractions of exosomes. Exiqon Services uses the miRCURY™ Exosome Isolation Kit to ensure a very high recovery of exosomes,. Following exosome isolation, our proprietary protocol for RNA Isolation ensures high quality microRNA.

Figure 1 The structure of an exosome. (Click to learn more)
The structure of an exosome. (Click to learn more)


Figure 2 The hypothesis of exosomal shuttling of microRNAs. (Click to learn more)
The hypothesis of exosomal shuttling of microRNAs. (Click to learn more)

 
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