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Optimized Quantitative Assessment of Enhancer RNA Stability in Mouse Embryonic Stem Cells

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Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D.

Fabiana Perocchi¹, Zhenyu Xu, Sandra Clauder-Münster

¹European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

Nucleic Acids Research

|September 28, 2007

Summary

Researchers developed a new method using actinomycin D (ActD) to remove experimental artifacts in transcriptome profiling. This technique accurately identifies true antisense transcription, improving our understanding of its cellular roles.

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In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Published on: May 5, 2023

Area of Science:

Genomics
Molecular Biology
Transcriptomics

Background:

Antisense transcription is increasingly detected in genomes, but its prevalence and significance are debated due to suspected experimental artifacts.
Distinguishing genuine antisense transcripts from artifacts is crucial for accurate genomic analysis.

Purpose of the Study:

To develop and validate a method for generating clean, genome-wide transcriptome profiles.
To differentiate true antisense transcripts from artifacts in gene expression studies.

Main Methods:

Utilized actinomycin D (ActD) during the reverse transcription step of RNA isolation.
Compared strand-specific hybridization signals from Saccharomyces cerevisiae tiling arrays with and without ActD treatment.
Analyzed the impact of ActD on sense and antisense transcript detection.

Main Results:

Actinomycin D (ActD) treatment effectively removed approximately 50% of detected antisense transcripts, indicating they were artifacts.
Sense transcript levels remained largely unaffected by ActD.
Around 200 genuine antisense transcripts were identified and unaffected by the treatment.

Conclusions:

The developed method using ActD enables cleaner transcriptome profiling by mitigating spurious second-strand cDNA synthesis artifacts.
This approach allows for a more accurate assessment of the extent and location of true antisense transcription.
Findings contribute to a better understanding of the functional roles of antisense transcripts in cellular processes.