Circulating miRNAs Sequencing (Cell-Free miRNA Seq)

At CD Genomics, we offer a comprehensive range of untargeted circulating miRNA detection and analysis services, utilizing our small RNA next-generation sequencing and data analysis platforms. Our services are designed to enable your extracellular miRNA research and biomarker discovery breakthroughs.

Overview

Since 2008, circulating miRNAs have been discovered in various extracellular human body fluids such as serum, plasma, saliva, urine, tears, etc. These circulating miRNAs are encapsulated in small membrane vesicles (e.g., exosomes, microparticles, and apoptotic bodies) or bound to proteins such as HDL. They play a crucial role in intercellular communication, as they are released into the bloodstream upon tissue damage or active secretion, and are transported to target cells to regulate gene expression and impact cellular function.

The stability of circulating miRNAs is a significant advantage. Unlike intracellular miRNAs, miRNAs in biofluids are protected from degradation by RNase enzymes due to their encapsulation or protein binding. They exhibit remarkable stability, allowing for long-term storage at room temperature and resistance to multiple freeze-thaw cycles and extreme pH conditions. Furthermore, circulating miRNAs show low variability and remain relatively stable over the lifespan of healthy individuals, making them potentially valuable as disease biomarkers.

Our team of experts is dedicated to delivering high-quality sequencing data and comprehensive analysis to uncover the potential of circulating miRNAs and exosome miRNAs in disease diagnostics, therapeutic response monitoring, and biomarker discovery.

Are you interested in our Exosome miRNA Sequencing Solution? Take a look at our website to know about cellular health, disease states, and more by analyzing exosomes and extracellular vesicles (EVs).

Project Workflow

Bioinformatics Analysis Pipeline

1. Read Alignment and miRNA Annotation

2. Quantification and Normalization

3. Differential Expression Analysis

4. Functional Analysis

5. Biomarker Identification

Case Studies

• • miRNAs in Osteoarthritis Research

    • Background

      Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation, affecting millions of people worldwide. MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression and play a crucial role in various biological processes, including OA pathogenesis. However, the comprehensive profiling of circulating miRNAs in different stages of OA remains limited.

      Key Challenge

      The key challenge was to conduct a comprehensive analysis of the circulating miRNome in patients with early and late-stage symptomatic radiographic knee OA. The goal was to identify unique miRNA signatures associated with each disease state and gain insights into the underlying mechanisms driving OA progression.

      Methods

      Ninety-one patients diagnosed with knee OA were recruited, comprising 41 patients with early-stage OA (Kellgren–Lawrence [KL] grade 0 or 1) and 50 patients with late-stage OA (KL grade 3 or 4). Plasma samples were collected from each participant, and miRNA libraries were prepared using miRNA Library Kit. The libraries were subjected to high-throughput sequencing using the Illumina NextSeq 550 platform. The obtained miRNA expression data were analyzed using statistical, bioinformatics, and computational biology approaches.

      Results

      The sequencing analysis revealed 215 differentially expressed miRNAs (FDR < 0.01) between early and late-stage OA samples. Among these, 97 miRNAs exhibited consistent alterations in expression (increase or decrease) in ≥85% of the early OA group compared to the median expression in the late OA group. By increasing the threshold to ≥95%, a panel of seven miRNAs was identified: hsa-miR-335-3p, hsa-miR-199a-5p, hsa-miR-671-3p, hsa-miR-1260b, hsa-miR-191-3p, hsa-miR-335-5p, and hsa-miR-543. Additionally, four novel miRNAs were found to be present in ≥50% of early OA samples and shared 27 predicted gene targets with the prioritized set of predicted gene targets from the 97 miRNAs, suggesting common underlying mechanisms.

      Workflow of sequencing of microRNAs isolated from blood plasma. (Ali et al. 2020)

Sample Requirements

RNA sample (concentration ≥ 200 ng/uL, quantity ≥ 1 ug)

1.8 ≤ OD260/280 ≤ 2.2, OD260/230≥2.0, RIN ≥ 6.5, 28S:18S≥1.0.

Please make sure that the RNA is not degraded.

Sample Storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with a sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

Reference:

1. Ali, Shabana A., et al. "Sequencing identifies a distinct signature of circulating microRNAs in early radiographic knee osteoarthritis." Osteoarthritis and Cartilage 28.11 (2020): 1471-1481.