RNA and cDNA sequencing

Explore transcriptomes at isoform level and generate new insights into RNA genomes with full-length nanopore RNA sequencing reads. Choose between cDNA sequencing, or unique to Oxford Nanopore, direct sequencing of native RNA. With no reverse transcription or amplification bias, direct RNA sequencing expands your analyses to include modified bases and poly-A tail length.


What is RNA sequencing?

RNA sequencing enables the analysis of RNA transcripts present in a sample from an organism of interest. The method provides a dynamic view of the cellular activity at the point of sampling, allowing characterisation of gene expression and identification of isoforms. However, accurate analysis of transcripts using traditional short-read sequencing technology can be challenging as RNA samples must be fragmented, copied into cDNA, amplified, sequenced in short sections, then reassembled, leading to potential multi-mapping — where short sequencing reads align to multiple locations.

With long nanopore reads, full-length transcripts can be sequenced end-to-end, facilitating their accurate, unambiguous analysis. Furthermore, nanopore sequencing does not require cDNA conversion or amplification, meaning base modifications are retained in the data. Researchers are using nanopore RNA sequencing to shed light on developmental biology, study the mechanisms underlying diseases such as cancers, and identify potential future disease-associated biomarkers. In addition to transcript analysis, RNA sequencing can also be applied to the study of RNA virus genomes.

Why nanopore technology for RNA and cDNA sequencing?

Using nanopore sequencing, you can:

  • Characterise and quantify full-length transcripts — up to single-cell resolution
  • Explore epigenetic modifications through direct RNA sequencing
  • Get faster access to results with real-time analysis and bespoke tools
  • Reduce bias with PCR-free protocols
  • Scale to your needs with Flongle, MinION, GridION, or PromethION

Find out more about RNA and cDNA sequencing

Learn more about the benefits of sequencing full-length transcripts with long nanopore reads and get best-practice recommendations for single-cell sequencing.

Experimental approach to RNA and cDNA sequencing

Sequencing kits are available for the preparation of both RNA and cDNA libraries. The Direct RNA Sequencing Kit allows native RNA molecules — including base modifications — to be sequenced directly. Enabling high-output sequencing of whole transcriptomes from low input amounts, the cDNA-PCR Sequencing Kit is ideal for identifying and quantifying full-length transcripts at the isoform level.

Direct RNA Sequencing Kit cDNA-PCR Sequencing Kit
  • PCR-free, full-length transcripts for unbiased isoform-level gene expression analysis
  • Detect RNA modifications
  • Fast and efficient workflow, saving time and effort
  • High-output sequencing of whole transcriptomes from low input amounts
  • Complete, isoform-level transcriptome characterisation — up to single-cell resolution
  • Simple workflow, highest output, and sample barcoding options
Preparation time 135 min ~225 min + PCR
Input recommendation 200–300 ng poly(A)+ RNA or 1 µg total RNA 10 ng poly(A)+ RNA or 500 ng total RNA
Fragmentation Optional Yes
Amplification No Yes
Barcode options In development PCR-cDNA Barcoding Kit (24 plex)
Adaptive sampling In development In development
Methylation included Yes No
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Sequencing devices

Which device for RNA and cDNA sequencing?

From powerful, portable Flongle and MinION devices, suitable for low-pass whole-transcriptome sequencing, to the flexible GridION and high-output PromethION platforms, ideal for isoform-level differential expression analysis — scale your RNA sequencing to match your specific research requirements.

Nanopore sequencing device output graph

Note: The Direct RNA Sequencing Kit and associated flow cells are not currently available for Flongle devices.

Recommended for RNA and cDNA sequencing

PromethION 2 devices

Offering the flexibility of two independent, high-output PromethION Flow Cells, the compact PromethION 2 devices bring the benefits of high-coverage, real-time nanopore sequencing to every lab. Ideal for low-cost access to highly accurate whole transcriptomes.

Analysis techniques for RNA and cDNA sequencing

Analysis solutions

Oxford Nanopore provides end-to-end bioinformatics workflows for the analysis of whole transcriptomes. The workflow wf-transcriptomes enables de novo or reference-guided transcript assembly from either cDNA or direct RNA reads, and provides differential gene expression and differential transcript usage analysis. For single-cell transcriptome analysis, the workflow wf-single-cell, a research pipeline, provides outputs including barcode and unique molecular identifier (UMI) tags, gene x cell expression matrices, and cell x transcript expression matrices. Both workflows are available in EPI2ME, Oxford Nanopore's intuitive software platform.

Find out more about analysing nanopore RNA and cDNA sequencing data.

Featured whole-transcriptome sequencing workflow

For high-throughput analysis of whole human transcriptomes, we recommend the following:

Inspiration for RNA and cDNA sequencing

Discover more about applying nanopore RNA and cDNA sequencing to your area of research.

Research areas


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