miRNA Sequencing Analysis Service

What is miRNA Sequencing Data Analysis

MicroRNAs (miRNAs) are small, non-coding RNA molecules, typically 20-24 nucleotides in length, that play essential roles in regulating gene expression. By binding to complementary sequences in messenger RNA (mRNA), miRNAs can induce mRNA degradation or inhibit translation, thereby modulating various biological processes such as development, differentiation, proliferation, and apoptosis. The biogenesis of miRNAs begins with the transcription of primary miRNAs (pri-miRNAs), which are processed into precursor miRNAs (pre-miRNAs) and subsequently into mature miRNAs. These mature miRNAs are integrated into the RNA-induced silencing complex (RISC), which mediates their binding to target mRNAs.

miRNA sequencing is a high-throughput technique used to profile the expression of miRNAs in a sample. This method involves extracting small RNA molecules, converting them into cDNA libraries, and sequencing them using next-generation sequencing (NGS) technologies. The resulting data provides comprehensive insights into miRNA expression levels, identifies novel miRNAs, and detects miRNA variants. miRNA sequencing is crucial for understanding miRNA roles in gene regulation, discovering biomarkers, and studying disease mechanisms, offering a powerful tool for both basic and translational research in various biological and medical fields.

Fig 1. miRNA biogenesis and the silencing mechanism (Dobrzycka M, et al. 2023).

Application of miRNA Sequencing Data Analysis

miRNA sequencing data analysis has diverse applications across various fields of biology and medicine:

• Diseases Research
• Developmental Biology
• Personalized Medicine
• Drug Development
• Biomarker Discovery

Advantages of CD Genomics

State-of-the-Art Equipment

Utilize advanced sequencing technology to ensure precise and reliable miRNA analysis, enabling high-throughput data generation for comprehensive miRNA profiling.

High Sensitivity and Specificity

Achieve exceptional detection accuracy in identifying low-abundance miRNAs, ensuring the capture of a broad spectrum of miRNA species with minimal false positives.

Rigorous Quality Control

Implement stringent quality control measures at every stage, from RNA extraction to data analysis, ensuring the integrity and reproducibility of miRNA sequencing results.

Efficient Adapter Trimming and Read Alignment

Employ optimized software tools for swift and accurate adapter removal and sequence alignment, minimizing data loss and enhancing mapping efficiency.

Robust Normalization and Differential Expression Analysis

Utilize advanced normalization techniques and statistical methods to accurately quantify and compare miRNA expression levels across different samples and conditions.

Expertise in Novel miRNA Discovery

Leverage extensive experience and cutting-edge algorithms to identify previously uncharacterized miRNAs, expanding the understanding of miRNA roles in various biological processes.

Integrated Analysis Capability

Our miRNA sequencing service at CD Genomics is enhanced by multi-omics analysis, integrating data from genomics, transcriptomics, and proteomics to provide a comprehensive understanding of miRNA functions and their regulatory networks.

CD Genomics Sequencing and Data Analysis Pipeline

Fig 2. Sequencing Pipeline of miRNA

Sample Submission Guidelines of Sequencing

Fig 3. Data Analysis of miRNA Sequencing

Bioinformatic Analysis Content

CD Genomics employs well-established academic algorithms to systematically classify and annotate miRNA, siRNA, piRNA, and other unknown small RNAs. We comprehensively investigate the regulatory functions of these small RNAs through base editing analysis, expression level analysis, and target gene prediction.

How It Works

Fig 4. How It Works.

miRNA Expression Distribution

Fig 5. Boxplot for each sample.

Fig 6. Density distribution.

Differential Expression Analysis

Fig 7. Correlation analysis between samples.

Fig 8. Volcano plot of significantly differentially expressed miRNA (Plus_vs_minus).

Fig 9. Statistics results of GO annotation for Plus_vs_minus.

Fig 10. Statistics results of KEGG enrichment for Plus_vs_minus.

Title: microRNA sequencing for biomarker detection in the diagnosis, classification and prognosis of Diffuse Large B Cell Lymphoma

Publication: Sci Rep

Main Methods: microRNA sequencing, small RNA-seq

Abstract: Diffuse Large B Cell Lymphoma (DLBCL) exhibits variable backgrounds, leading to heterogeneous patient outcomes, with 40% experiencing refractory disease or relapse. Our study aimed to identify a set of miRNAs useful as biomarkers for DLBCL diagnosis, classification, prognosis, and treatment response, and to elucidate the role of deregulated miRNAs in DLBCL pathogenesis. Analyzing miRNA expression in 78 DLBCL patients and 17 controls using small RNA sequencing, we identified new miRNA signatures and their potential mechanisms in DLBCL. Our findings highlight the significant role of miRNAs in DLBCL.

Main Research Results:

MiRNAs deregulated in DLBCL: MiRNA sequencing analysis identified 1,584 miRNAs, with principal component analysis (PCA) showing distinct clustering of patient and control samples. Comparing 78 DLBCL samples with 17 controls revealed 146 miRNAs with significant expression differences; 122 were upregulated and 24 downregulated.

MiRNAs as prognostic biomarkers in DLBCL and their impact on survival: Comparing miRNA expression at diagnosis between 50 patients with long-term remission and 16 who relapsed within 10 years revealed seven miRNAs upregulated in remission and three in relapse. Patient survival curves showed that high miR-370-3p expression was linked to better 5-year progression-free survival (PFS) (PFS p-value = 0.025). However, Cox multivariate analysis indicated miR-370-3p was not independently associated with PFS when considering IPI and subtype.

Fig 11. Survival analysis of miR-370-3p.

miRNA-mRNA interaction network analysis:122 upregulated miRNAs in DLBCL were found to have 482 validated targets, and 24 downregulated miRNAs had 375 targets. Integrating this with the GSE56315 dataset, we found 138 genes highly expressed in DLBCL targeted by downregulated miRNAs and 76 downregulated genes targeted by upregulated miRNAs. Pathway enrichment analysis revealed significant pathways affected by deregulated miRNAs, including the FoxO signaling, Receptor Tyrosine Kinases, and PI3K-Akt pathways.

Fig 12. Genes of pathways in cancer targeted by downregulated and upregulated microRNAs

Conclusion:

This study used next-generation sequencing to identify miRNA signatures relevant to DLBCL, suggesting their potential for diagnosis and therapy. Prospective studies are needed to validate these findings. Key mechanisms were explored, with miR-182-5p notably impacting the PI3K/AKT pathway in DLBCL pathogenesis.

1. How is miRNA sequenced?

MiRNA sequencing involves isolating small RNAs from a sample, converting them into cDNA libraries, and sequencing using NGS platforms. The resulting sequences are then analyzed to identify and quantify miRNAs, providing insights into their expression levels and potential roles in various biological processes.

2. What is the difference between small RNA and miRNA?

Small RNAs (sRNAs) constitute a diverse group of non-coding RNA molecules, generally ranging from 20 to 30 nucleotides in length, that are integral to regulating gene expression and maintaining genomic stability. This category includes miRNAs, small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs).

miRNAs represent a specific class of sRNAs, typically 20 to 22 nucleotides long. They primarily function by binding to complementary sequences on mRNA molecules, leading to either mRNA degradation or inhibition of translation.

It is important to note that while all miRNAs are classified as small RNAs, not all small RNAs fall under the miRNA category. The term "sRNA" encompasses a range of RNA molecules, each with distinct regulatory functions.

3. How long is miRNA sequencing?

MicroRNA (miRNA) sequencing typically involves sequencing short RNA molecules, usually around 20-22 nucleotides in length, to analyze their expression and regulatory functions.

4. Should you submit total RNA or isolated miRNA for miRNA sequencing?

To avoid miRNA loss during purification, submit total RNA rather than small RNAs. While we can sequence isolated miRNA, success isn't guaranteed due to small RNA extraction efficiency. We highly recommend monitoring the extraction process and providing the results to us for better sequencing library preparation.

5. What is bioinformatics analysis of miRNA sequencing data?

Bioinformatics analysis of miRNA sequencing data involves processing raw sequencing reads, aligning them to a reference genome, and identifying known and novel miRNAs. This analysis quantifies miRNA expression levels, detects differentially expressed miRNAs across conditions, and predicts their target genes. Further steps include functional enrichment analysis to understand the biological roles of miRNAs and their involvement in regulatory networks, contributing to insights into gene regulation and disease mechanisms.