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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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Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

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3' MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage.

Stefaniya Boneva¹, Anja Schlecht¹, Daniel Böhringer¹

¹Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Laboratory Investigation; a Journal of Technical Methods and Pathology

|May 30, 2020

Summary

3' massive analysis of cDNA ends (MACE) RNA-sequencing offers comprehensive transcriptome profiling from FFPE archival samples. This method shows minimal bias and is suitable for analyzing stored tissues, unlike standard RNA-sequencing.

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Area of Science:

Molecular Biology
Genomics
Bioinformatics

Background:

Standard RNA-sequencing (RNA-Seq) and 3' massive analysis of cDNA ends (MACE) RNA-sequencing are powerful tools for transcriptome analysis.
Formalin-fixed paraffin-embedded (FFPE) samples are valuable archival resources but pose challenges for molecular analysis.

Purpose of the Study:

To compare the efficacy of MACE RNA-sequencing against standard RNA-Seq for fresh tissue analysis.
To evaluate the transcriptome profiling capabilities of MACE RNA-sequencing on FFPE archival human samples.
To assess the impact of FFPE processing and storage time on MACE RNA-sequencing results.

Main Methods:

Fresh conjunctival tissue samples were analyzed using both standard RNA-Seq and MACE RNA-Seq.
FFPE-processed conjunctival tissue was analyzed by MACE RNA-Seq to assess FFPE impact.
Transcriptome profiling was performed on 24 FFPE conjunctival samples with varying storage times.

Main Results:

MACE and standard RNA-Seq detected a high number of common transcripts in fresh tissue, with MACE showing less length bias.
FFPE processing had negligible effects on MACE sequencing results, and library diversity was maintained during long-term storage.
While raw read counts decreased non-linearly with storage time (especially within the first 1000 days), the number of detected transcribed genes remained independent of FFPE storage duration.

Conclusions:

MACE RNA-sequencing is a robust method for comprehensive transcriptome profiling of FFPE archival samples.
The technology is less susceptible to transcript length bias compared to standard RNA-Seq.
MACE RNA-sequencing presents a valuable opportunity for leveraging historical FFPE tissue collections for genetic research.