RNA Sequencing
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Small RNA and miRNA sequencing

Profile and discover small RNA and microRNA species with next-generation sequencing

Introduction to small RNA sequencing

Small RNAs are short, non-coding RNAs that play an important role in regulating gene expression through gene silencing and post-transcriptional activity. Small RNA sequencing (small RNA‑Seq) delivers sensitive, single‑base resolution insights into microRNAs (miRNAs) and other small noncoding RNAs involved in gene regulation, development, and disease. Whether you’re exploring circulating miRNA biomarkers, characterizing miRNA isoforms (isomiRs), or profiling diverse small RNA species, Illumina sequencing provides accurate, high‑throughput measurement across sample types and study designs.

Advantages of small RNA and miRNA sequencing

Small RNA sequencing

Small RNA-Seq enables comprehensive analysis of short, regulatory RNA molecules that are not effectively captured by traditional mRNA‑focused sequencing approaches. By leveraging next‑generation sequencing (NGS), small RNA‑Seq provides an unbiased view of small RNA expression across diverse sample types.

Key advantages include:

  • Simultaneous profiling of multiple small RNA classes to measure miRNAs alongside other regulatory small RNAs in a single assay
  • Sequence‑based detection without prior probe design to identify known and novel small RNAs without relying on predefined probes or primers, enabling discovery‑driven research
  • Single‑base resolution across short RNA species to capture precise sequence and length information critical for studying RNA biogenesis, processing, and regulation
  • High sensitivity across a wide dynamic range to detect both highly expressed and low‑abundance small RNAs across biological conditions and sample inputs
  • Compatibility with challenging samples to analyze fragmented or low‑input RNA due to the naturally short length of small RNAs (sample compatibility varies depending on the library prep kit)

miRNA sequencing

miRNA-Seq focuses on the accurate measurement of microRNAs that function as key regulators of post‑transcriptional gene expression to influence development, disease progression, cellular pathways, and more. Within small RNA‑Seq workflows, miRNA sequencing enables deep biological insights into miRNA‑driven regulatory networks.

Key advantages include:

  • Accurate quantification of mature miRNA expression to measure relative miRNA abundance with high precision across samples, conditions, and time points
  • Detection of isomiRs and sequence variants to resolve miRNA isoforms differing by single bases or length, which may have distinct biological functions and regulatory targets
  • Discovery of previously unannotated miRNAs to identify novel miRNA species and tissue‑ or condition‑specific expression patterns
  • Suitability for low‑input samples to enable research with liquid biopsy, plasma, serum, and other limited or degraded samples (sample compatibility varies depending on the library prep kit)
  • Complementarity with other gene expression studies to integrate miRNA expression data with mRNA or multiomic datasets to reveal regulatory relationships and biological pathways
RNA sequencing methods guide cover. Close up image of a female scientist pipetting clear liquid into tube; NextSeq 1000/2000 reagent cartridge and sample plate on the lab bench.

RNA sequencing methods guide

This guide provides solutions for profiling RNA, from targeted panels to the whole transcriptome. Illumina RNA-Seq workflows integrate library prep, sequencing, and data analysis to support transcriptome research.

Sequencing workflows

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Library prep
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Sequencing
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Data analysis, storage, & insights
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Analyze and visualize small RNA-Seq data

DRAGEN secondary analysis is a powerful suite of software tools that provides accurate, comprehensive, and efficient analysis of NGS data. The DRAGEN miRNA application enables accurate alignment and quantification of miRNA and other small RNAs from total RNA extracted from diverse sample types, including FFPE tissue, blood, and cell cultures.

Illumina Connected Multiomics is a powerful, scalable analysis environment for interpreting and visualizing multiomic data. Illumina Connected Multiomics can analyze miRNA-Seq data to find differentially expressed miRNAs and integrate the results with differentially expressed messenger RNAs quantified in the same tissues. This can provide an overall picture of how miRNAs drive widespread changes in cellular function.

Redefining NGS in cancer research

Learn how NGS can be used to characterize the proteome, epigenome, non-coding RNAs, and small RNAs.

View webinar

FAQ

Small RNA sequencing is used to profile short noncoding RNA molecules, such as microRNAs (miRNAs), that regulate gene expression and cellular pathways. It is widely applied in biomarker discovery, disease research, and functional genomics.

Small RNA sequencing captures the full spectrum of small RNA species, whereas miRNA sequencing focuses only on miRNA.

Messenger RNA sequencing (mRNA‑Seq) measures long, protein‑coding transcripts, while small RNA‑Seq targets short, non-coding RNAs that regulate post‑transcriptional gene expression. These approaches as a whole provide complementary views of gene regulation.

Illumina miRNA Prep is optimized for 1-500 ng of total RNA.

Single‑end sequencing is recommended for small RNA‑Seq and miRNA sequencing and is sufficient for accurate alignment and quantification. Paired‑end sequencing offers no advantage for small RNA libraries because the full small RNA insert is captured in a single read.

Learn more about paired-end vs single-read sequencing

Yes, small RNAs are too small to be captured by total RNA and mRNA library preparation kits. Library fragments generated from such small RNAs are not faithfully retained during bead clean-up steps.

Learn more about small RNA with our reference material resource.

Common types of small RNA include micro RNA (miRNA), small interfering RNA (siRNA), and Piwi-interacting RNA (piRNA).

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Related applications

Cancer research with RNA-Seq

Study cancer gene expression and transcriptome changes and identify novel cancer transcripts with RNA sequencing.

Complex disease studies

NGS-based profiling enables rapid, high-throughput analysis of the miRNA transcriptome, and can provide insights into complex disease-related processes and mutational events.

Epigenetic analysis

Illumina offers a broad portfolio of epigenetics tools for investigators interested in studying epigenetic modifications and gene regulation.

Additional resources

Exploring non-coding RNA in cancer

This work illustrates how transcriptome‑wide RNA‑Seq reveals the regulatory complexity of long non-coding RNA, and underscores the importance of RNA‑based insights in cancer and disease research.

Push-button informatics

Illumina bioinformatics tools make it easier to manage, analyze, and interpret large amounts of complex genomic data.

RNA sequencing considerations

This article reviews considerations for various RNA-Seq experiment types and offers resources to help with study design.

Speak to a specialist

Talk to an expert to learn more about small RNA-Seq solutions.