Exosomal mRNA Sequencing Service

Q: Q1: Can Exosomal mRNA Sequencing detect all types of mRNA transcripts, including rare or low-abundance ones?

A: Yes. We use the Illumina NovaSeq 6000 which covers low-abundance exosomal mRNA down to 1 copy per cell. Unlike small RNA sequencing (which focuses on high-abundance miRNAs), our high read depth ensures even rare mRNA transcripts (e.g., tissue-specific transcription factors) are detected.

Q: Q2: What's the difference between strand-specific and non-strand-specific exosomal mRNA sequencing, and why do you use the former?

A: Strand-specific sequencing preserves the direction of mRNA transcription (sense vs. antisense), while non-strand-specific sequencing cannot distinguish between the two. This is vital for exosomal mRNA analysis because antisense mRNA in exosomes often regulates the expression of sense mRNA (e.g., by blocking translation). Without strand information, you might misinterpret antisense transcripts as functional sense mRNA, leading to incorrect conclusions. Our strand-specific workflow avoids this issue, making it ideal for studying transcriptional regulation.

Q: Q3: If my samples are from non-model species (e.g., agricultural crops or rare animals) with no reference genome, can you still perform exosomal mRNA sequencing?

A: Absolutely. For non-model species, we provide de novo transcriptome assembly. This process constructs a reference transcriptome directly from your exosomal mRNA sequencing data. It then enables the identification of mRNA transcripts, alternative splicing events, and differential expression, all without needing a pre-existing genome.

Q: Q4: How long does it take to get results from exosomal mRNA sequencing, and can you expedite the process for time-sensitive projects?

A: The standard turnaround time is 3-4 weeks from sample receipt to report delivery, which includes exosome isolation (if needed), mRNA extraction, sequencing, and in-depth analysis (e.g., alternative splicing, fusion gene detection). For urgent projects (e.g., clinical trial biomarker validation), we offer an expedited service that shortens the timeline to 2-3 weeks by prioritizing your samples in our workflow and streamlining analysis steps. Note that expediting is feasible because our mRNA-specific protocols are optimized for efficiency-unlike small RNA sequencing, which has fewer complex analysis steps (e.g., no fusion gene detection) to accelerate.

Q: Q5: Can you compare exosomal mRNA profiles between different sample groups (e.g., healthy vs. disease) and highlight key pathways involved?

A: Definitely. Our analysis includes group-wise differential expression screening (using DESeq2/edgeR) to identify mRNA transcripts with significant expression changes (fold change ≥2, p<0.05). We then map these differential mRNAs to GO terms (e.g., "cell proliferation") and KEGG pathways (e.g., "PI3K-Akt signaling") to reveal biological processes driving the difference. For example, in a study of Alzheimer's disease, we compared cerebrospinal fluid exosomal mRNA between patients and controls, finding differential mRNAs enriched in "amyloid-beta clearance" pathways. This pathway-level analysis is more impactful for mRNA sequencing (which targets functional genes) than for small RNA sequencing (which focuses on regulatory RNAs with indirect pathway links).

Exosomal mRNA is a critical cargo in exosomes, carrying full-length genetic information of functional genes from donor cells to recipient cells, directly reflecting transcriptional activity. Creative Biostructure's exosome mRNA sequencing service captures full-length mRNA transcripts, enabling comprehensive analysis of gene expression changes, alternative splicing events, and fusion genes in exosomes. Powered by advanced Illumina sequencing technology and optimized mRNA-specific workflows, we deliver high-quality data to our clients.

Why is Exosomal mRNA Sequencing Service Important?

Exosomal mRNA sequencing service is critically important for both basic research and clinical applications, as it opens a unique window into cellular activity and intercellular communication. Exosomes carry cell-type-specific mRNA cargo that reflects the functional state of their parent cells, offering a non-invasive method to access biologically significant information that is often inaccessible through traditional methods. By employing high-throughput sequencing, this service enables the comprehensive profiling of these mRNA molecules, allowing researchers to identify novel biomarkers for early disease diagnosis, monitor treatment response in real time, and uncover fundamental mechanisms underlying disease progression. In precision medicine, it provides particularly valuable insights for understanding cancer biology, neurodegenerative disorders, and developing targeted therapies, ultimately bridging the gap between basic science and clinical practice.

Discovery of circulating exosomal mRNA biomarkers for prostate cancer detection. Figure 1. Circulating exosomal mRNA profiling identifies novel signatures for the detection of prostate cancer. (Ji J, et al., 2021)

Our Exosomal mRNA Sequencing Service

We adopt the Illumina NovaSeq 6000 high-throughput sequencing platform, tailored for exosomal mRNA's characteristics (longer length, lower abundance vs. small RNA):

Technology	Description
PolyA+ Enrichment Technology	Uses magnetic beads with oligo(dT) probes to specifically capture mRNA (excluding rRNA, tRNA, and small RNA/miRNA), achieving ≥98% rRNA removal rate and ensuring mRNA purity.
Strand-Specific Library Construction	Preserves the transcriptional direction of mRNA (sense/antisense), which is essential for analyzing antisense mRNA regulation and avoiding false positive results from reverse strands.
High Read Depth Configuration	30-50 million clean reads per sample, covering low-abundance exosomal mRNA (down to 1 copy per cell) and supporting accurate detection of alternative splicing isoforms.

Workflow of Exosomal mRNA Sequencing Analysis

1

Exosome Isolation

For raw samples (e.g., serum, cell supernatants), use "ultracentrifugation + size-exclusion chromatography" to isolate exosomes. This method effectively removes lipoprotein contamination (a major interference for mRNA extraction), which is more precise than the simple centrifugation used in small RNA sequencing.

2

mRNA-Specific Extraction

Use RNase-free kits with PolyA+ magnetic beads to extract mRNA from exosomes; add DNase I to eliminate genomic DNA contamination (critical for avoiding false positive gene expression results).

3

Strand-Specific Library Prep

- Fragment mRNA into 200-300 bp segments (optimized for mRNA length, not small RNA's 18-30 bp).
- Synthesize first-strand cDNA with reverse transcriptase (labeled with dUTP to mark the reverse strand).
- Digest the reverse strand with UDG enzyme to retain only the forward strand, then add Illumina adapters to construct libraries.

4

High-Throughput Sequencing

Load libraries onto the Illumina NovaSeq 6000 platform for paired-end sequencing (2×150 bp), ensuring sufficient coverage for alternative splicing detection.

5

mRNA-Focused Data Analysis

- Basic Analysis: FastQC (quality control of raw reads), STAR (genome alignment with splice junction recognition), HTSeq (gene/isoform quantification);
- Advanced Analysis: DESeq2/edgeR (differential gene expression screening), rMATS (alternative splicing event identification), STAR-Fusion (fusion gene detection), GO/KEGG/GSEA (functional enrichment of differential genes).

Process for exosomal mRNA sequencing analysis. Figure 2. Workflow of exosomal mRNA sequencing analysis. (Creative Biostructure)

Supported Sample Range

Raw Samples:

Serum/plasma: ≥200 μL, no hemolysis (hemoglobin degrades mRNA).
Cell culture supernatants: ≥10 mL, freshly collected (avoid long-term storage).
Cerebrospinal fluid: ≥100 μL, stored at -80°C immediately.
Urine: ≥500 μL, add RNA protection agent at collection.

Final Delivery

Clients receive a comprehensive, publication-ready report including:

QC Report: mRNA integrity (RIN value), rRNA removal rate, library concentration/uniformity, sequencing Q30 score, and alignment rate.
Raw & Processed Data: FASTQ files (raw reads), BAM files (aligned reads), gene/isoform expression matrix (TPM/FPKM values).
Advanced Analysis Results:
- Differential gene list (fold change ≥2, p<0.05) with volcano plots and heatmaps.
- Alternative splicing events (skipped exons, alternative 5'/3' splice sites) with rMATS statistics.
- Fusion gene list (if detected) with breakpoint coordinates and annotation.
Functional Interpretation: GO/KEGG enrichment analysis (focused on biological processes like "cell proliferation" "metabolic pathway") and GSEA analysis of differential gene sets.

Applications of Exosomal mRNA Sequencing

Alternative Splicing in Disease: Study how exosomal mRNA splicing variants drive disease.
Fusion Gene Biomarker Discovery: Identify exosomal fusion mRNA as cancer biomarkers.
Transcriptional Response to Therapy: Monitor changes in exosomal mRNA expression during treatment.
Tissue-Specific Communication Research: Analyze organ-specific mRNA in exosomes to study inter-organ communication.

Advantages of Our Exosomal mRNA Sequencing Profiling Services

mRNA-Specific Capture: PolyA+ enrichment ensures no small RNA/miRNA interference, focusing on functional gene transcripts.
Splicing & Fusion Detection: Unique ability to analyze alternative splicing and fusion genes.
High Transcriptional Resolution: Strand-specific libraries reveal antisense mRNA regulation, providing deeper insights into transcriptional mechanisms.
Reliable Low-Abundance Detection: 30-50 million reads cover low-abundance exosomal mRNA, avoiding missed detection of key functional genes.
Expert Data Interpretation: Our team of computational biologists specializes in mRNA analysis, not just general sequencing, ensuring accurate interpretation of splicing and fusion results.

Case Study

Case: Biomarker Discovery and Clinical Relevance via Serum Exosomal mRNA-seq in Non-Hodgkin Lymphoma (NHL)

Background

The clinical utility of mRNA cargo in exosomes is unclear, although exosomes have potential as non-invasive biomarkers. This study aimed to investigate the feasibility of exosomal mRNA sequencing for monitoring disease status and predicting outcomes in non-Hodgkin lymphoma (NHL) patients.

Methods

Isolation of exosomes by ultracentrifugation.
Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).
Characterization of exosomes.
Sequencing of exosomal mRNA.

Conclusion

Exosomal mRNA sequencing was performed successfully in 26 cases (79%, 26/33), whereas the remaining seven cases were not completed due to their small amount of RNA. The exosomal mRNA sequencing of DLBCL showed gene expression profiles consistent with activated B-cell-like and germinal center type.

Characterization of the exosomal mRNA expression landscape in non-hodgkin lymphoma patients. Figure 3. Exosomal mRNA expression profile in patients with non-Hodgkin lymphoma heatmap of mRNA expression levels for selected genes at baseline. (Bang Y H, et al., 2022)

Creative Biostructure is committed to providing clients with highly effective solutions. Whether in exploring disease mechanisms, discovering biomarkers, or conducting translational medicine research, we are dedicated to offering reliable support to help clients make progress in the field of exosome research. Please cntact us for more detailed information. We will be happy to assist you.

References

Bang Y H, Shim J H, Ryu K J, et al. Clinical relevance of serum-derived exosomal messenger RNA sequencing in patients with non-Hodgkin lymphoma. Journal of Cancer. 2022, 13(5): 1388.
Ji J, Chen R, Zhao L, et al. Circulating exosomal mRNA profiling identifies novel signatures for the detection of prostate cancer. Molecular cancer. 2021, 20(1): 58.

Frequently Asked Questions

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