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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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Reproducible and sensitive micro-tissue RNA sequencing from formalin-fixed paraffin-embedded tissues for spatial gene

Hiroko Matsunaga¹, Koji Arikawa¹, Miki Yamazaki^2,3

¹Research Organization for Nano and Life Innovation, Waseda University, Tokyo, Japan.

Scientific Reports

|November 14, 2022

Summary

A new method enables spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues, even after years of storage. This technique allows detailed gene expression mapping in FFPE tissue sections for clinical research.

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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

Area of Science:

Molecular Biology
Genomics
Pathology

Background:

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable for long-term storage and clinical research.
However, FFPE processing can damage RNA, limiting gene expression analysis.
Fresh-frozen tissues offer better RNA quality but lack the preservation benefits of FFPE.

Purpose of the Study:

To develop a sensitive RNA-sequencing (RNA-seq) method for spatial transcriptome analysis of FFPE tissues.
To assess the feasibility of analyzing gene expression in micro-dissected FFPE tissue samples.
To evaluate the method's utility in clinical FFPE specimens, including those stored for extended periods.

Main Methods:

Proposed a novel FFPE micro-tissue RNA-seq method.
Combined micro-punching of tissue sections (100 μm diameter) with direct RNA-seq library construction.
Evaluated the method on mouse liver FFPE tissues (2 years post-fixation) and clinical lung cancer FFPE specimens (6 years post-fixation).

Main Results:

Detected approximately 7000 genes in micro-punched FFPE tissue spots (10 μm thickness).
Achieved gene detection levels comparable to purified total RNA from equivalent cell numbers.
Successfully identified characteristic gene expression in the tumor microenvironment of clinical FFPE lung cancer samples.

Conclusions:

The developed FFPE micro-tissue RNA-seq method is sensitive and effective for spatial gene expression analysis.
This technique is suitable for analyzing FFPE tissues, even those stored for many years in medical facilities.
Enables detailed study of the tumor microenvironment in FFPE samples, supporting clinical applications.