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Small RNA Sequencing (sRNA‑seq)

Small RNA sequencing (sRNA-seq) is a high-throughput next-generation sequencing (NGS) method used to identify, quantify, and characterize small RNA molecules, including miRNAs, siRNAs, piRNAs, and other regulatory RNAs. This approach enables both the detection of known small RNAs and the discovery of novel small RNA species, providing insights into their roles in gene regulation, development, stress response, and disease biology.


Novogene’s small RNA-seq service offers highly sensitive, high resolution profiling of diverse small RNA populations in a single assay. Supported by a robust in-house bioinformatics pipeline, the workflow enables accurate differential-expression analysis, discovery of novel small RNAs, evaluation of structural or sequence alterations, and comprehensive characterization of miRNA expression patterns across experimental conditions.

Benefits of Novogene Small RNA Sequencing

Dual Sample Type CompatibilityDual Sample Type Compatibility
Dual Sample Type Compatibility

Supports total RNA and exosomal RNA inputs, enabling comprehensive exploration of intracellular and extracellular small RNA dynamics across multiple biological compartments.

Novel Small RNA DiscoveryNovel Small RNA Discovery
Novel Small RNA Discovery

Detects previously unannotated small RNA species, expanding understanding of miRNAs, siRNAs, piRNAs, and other regulatory RNAs involved in diverse cellular processes.

Disease Associated Expression Profiling
Disease Associated Expression Profiling

Identifies differentially expressed small RNAs linked to cancer, neurological disorders, metabolic diseases, and other pathologies—supporting biomarker discovery and therapeutic target identification.

Regulatory Mechanism Decoding
Regulatory Mechanism Decoding

Clarifies post-transcriptional gene regulation mediated by small RNAs, providing insight into RNA-RNA and RNA-mRNA interaction networks and the molecular pathways they control.

Applications of Small RNA Sequencing

Small RNA sequencing (small RNA-seq) enables high-resolution profiling of regulatory small RNAs, offering powerful applications in both research and translational studies.

Precise Small RNA Profiling

Quantify known small RNAs (e.g., miRNAs, siRNAs, piRNAs) and discover novel small RNA molecules with high sensitivity and accuracy.

Disease Biomarker Discovery

Identify diagnostic and prognostic biomarkers for cancer, metabolic disorders, cardiovascular diseases, and infectious diseases by detecting small RNA expression changes.

Regulatory Mechanism Elucidation

Characterize post-transcriptional gene regulation mediated by small RNAs, revealing how miRNAs and other small RNAs control mRNA stability, translation, and network level regulatory pathways.

Therapeutic Target Exploration

Predict and validate small RNA–mRNA interactions to support drug development, target prioritization, and personalized medicine initiatives, including the development of miRNA-based therapeutics.

Specifications

Sample Requirements

Sample amounts are listed for reference only. Download the Sample Submission Guidelines to learn more. For detailed information, please contact us with your customized requests.

Library TypeSample TypeAmountRNA Integrity Number (Agilent 5400)Purity (NanoDrop)
Small RNA LibraryTotal RNA≥ 1000 ngAnimal ≥ 7.5, Plant ≥ 7, with smooth baselineOD260/280≥ 2.0;
OD260/230≥ 2.0;
no degradation, no contamination
Exosomal Small RNA LibraryExosomal RNA≥ 5 ngFragments between
25-200nt, FU>10

Sequencing and Analysis

Recommended data outputs and analysis contents displayed are for reference only. For detailed information, please contact us with your customized requests.

Sequencing PlatformIllumina NovaSeq System
Read LengthSingle-end 50 bp
Recommended Sequencing Depth≥ 10 million read pair per sample
Data qualityGuaranteed ≥ 85% bases with Q30 or higher
Standard Analysis (miRNA)• Data QC (adapter & low quality filtering)
• Length distribution summary
• Read mapping to reference genome
• Small RNA annotation (known miRNA, non coding RNA, repeats, exon/intron)
• Novel miRNA prediction
• miRNA base editing analysis
• miRNA family analysis
• miRNA expression quantification
Standard Analysis (siRNA; virus related samples only)• Data quality control
• Length distribution summary
• Sequence assembly
• Contig classification & annotation
• Mapping to host, NR, NT, and viral reference databases
• Virus species candidate summary
Advanced Analysis (miRNA–mRNA Association)• Integrated analysis of differentially expressed miRNA & mRNA
• Target gene prediction
• Clustering analysis
• GO enrichment / KEGG pathway enrichment
• Protein–protein interaction analysis (PPI)
Advanced Analysis (piRNA)• piRNA identification & quantification
• Base bias & chromosome distribution
• Source gene analysis
• Functional enrichment
• piRNA cluster analysis
• Differential expression (with biological replicates)

Novogene Small RNA Sequencing Workflow

Novogene’s small RNA-seq workflow follows a standardized, QC-driven process to ensure accurate and reproducible small RNA profiling. After sample preparation and RNA quality assessment, 3′- and 5′-adaptors are ligated to small RNA molecules, followed by first-strand cDNA synthesis and PCR enrichment to generate double-stranded cDNA libraries. Sequencing is performed on the Illumina platform, and data are processed through Novogene’s established bioinformatics pipeline for small RNA identification, quantification, and novel small RNA discovery. Customized analysis options are available upon request.

Novogene Small RNA Sequencing Workflow

Resources

Demo Result

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Length Distribution of Total small RNA

Small RNA molecules typically range from 18–40 nucleotides (nt). Examining their length distribution helps assess the composition of the small RNA population and provides insight into the relative abundance of different small RNA classes.

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Repeat Sequence Alignment

Small RNAs are aligned to predicted repeat sequences, and reads originating from repetitive regions are filtered out. Summary statistics are then generated to show the distribution of repeat‑derived small RNAs and the total number of retained small RNAs.

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miRNA TPM Distribution

TPM-based density distribution illustrates miRNA expression patterns across samples. In the plot, the x-axis represents sample names, and the y-axis shows log10(TPM + 1) values for each miRNA.

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Cluster Analysis of Differentially Expressed miRNAs

Clustering analysis groups miRNAs with similar expression profiles, enabling the identification of shared expression patterns across different experimental conditions.

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Scatter Plot of Target Gene Candidates

This plot visualizes enriched Gene Ontology (GO) terms associated with predicted miRNA target genes. The x-axis displays Gene Ontology (GO) terms, while the y-axis indicates their statistical significance.

Webinar

Novogene’s Comprehensive Introduction to Non-coding RNA Sequencing Services and Ultra-low Input RNA Sequencing Pipeline

Kick off the new academic year by joining our webinar to deepen your understanding of non-coding RNA sequencing and bioinformatics analysis. Discover insights into the regulatory roles of ncRNAs in gene expression and learn about our newly launched ultra-low input RNA pipeline, designed to provide comprehensive data on both mRNA and ncRNAs from a single library preparation, even with limited sample amounts.

(Novogene’s Comprehensive Introduction to Non-coding RNA Sequencing Services and Ultra-low Input RNA Sequencing Pipeline)

Frequently Asked Questions

What types of small RNAs can small RNA-seq detect?

Small RNA-seq can profile a wide range of regulatory small RNAs, including miRNAs, siRNAs, piRNAs, tRNA-derived fragments, and other non-coding RNA species. It supports both known small RNA annotation and novel small RNA discovery.

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