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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

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Related Experiment Video

Updated: May 10, 2025

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Published on: May 5, 2018

Single-nucleus total RNA sequencing of formalin-fixed paraffin-embedded samples using snRandom-seq.

Ziye Xu^1,2, Yuexiao Lyu¹, Haide Chen¹

¹Department of Laboratory Medicine of The First Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of Medicine, Hangzhou, China.

Nature Protocols

|April 25, 2025

Summary

snRandom-seq enables single-nucleus RNA sequencing from challenging formalin-fixed paraffin-embedded (FFPE) tissues. This novel method enhances RNA detection and offers a 4-day protocol for analyzing archived clinical specimens.

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Last Updated: May 10, 2025

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

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Published on: May 5, 2018

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Published on: June 8, 2020

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Published on: August 25, 2020

Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

Formalin-fixed paraffin-embedded (FFPE) tissues are a rich source of clinical data but difficult for single-nucleus RNA sequencing.
Existing high-throughput single-cell/single-nucleus RNA sequencing (sc/snRNA-seq) methods face challenges with FFPE sample quality and RNA integrity.

Purpose of the Study:

To develop and validate a novel method for single-nucleus RNA sequencing (snRNA-seq) specifically optimized for FFPE tissues.
To overcome limitations in RNA capture and detection in archived clinical samples.

Main Methods:

Developed snRandom-seq, a droplet- and random primer-based technology for FFPE snRNA-seq.
Protocol includes single-nucleus isolation, in situ DNA blocking, reverse transcription, dA tailing, barcoding, and library preparation.
Utilizes random primers for comprehensive total RNA capture.

Main Results:

snRandom-seq achieved a low doublet rate of 0.3%.
Demonstrated increased RNA coverage and enhanced detection of non-coding and nascent RNAs compared to other methods.
The entire protocol is completed within 4 days.

Conclusions:

snRandom-seq is a powerful and efficient platform for snRNA-seq analysis of FFPE clinical specimens.
This technology expands the utility of archived tissues for studying complex biological systems and diseases.
Enables deeper insights into cellular heterogeneity and molecular mechanisms from historical patient cohorts.