Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D
Open accessAbstract
Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthesis of second-strand cDNA during reverse transcription reactions. Strand-specific hybridization signals obtained from Saccharomyces cerevisiae tiling arrays were compared between samples prepared with and without ActD. Use of ActD removed about half of the detectable antisense transcripts, consistent with their being artifacts, while sense expression levels and about 200 antisense transcripts were not affected. Our findings thus facilitate a more accurate assessment of the extent and position of antisense transcription, towards a better understanding of its role in cells.
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5 μM Actinomycin D was kept during collection and enzymatic dissociation of the tissue to preserve transcriptional state and prevent artificial induction of stress-response genes during tissue dissociation.
“Note that 5 μM ActD was kept during collection and enzymatic dissociation of the tissue to preserve transcriptional state and prevent artificial induction stress-response genes during tissue dissociation (Marsh et al, 2022; Perocchi et al, 2007).”
From APOE from astrocytes restores Alzheimer’s Aβ-pathology and DAM-like responses in APOE deficient microglia (2024) · cites this paper as Perocchi et al, 2007
The Introduction (page unavailable) states that ActD inhibits transcription, which supports the general rationale for limiting new transcription. But the paper uses ActD during reverse transcription of yeast RNA, not at 5 μM during tissue collection or enzymatic dissociation; it does not show prevention of dissociation-induced stress-response genes. Materials and Methods (page unavailable) instead specifies 6 μg/ml ActD during cDNA synthesis. (LLM, conf=0.85)
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The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing
Nagalakshmi, U., Wang, Z., Waern, K., Shou, C., Raha, D., Gerstein, M., & Snyder, M. (2008) · Science · doi:10.1126/science.1158441 · Cited by 2,479 · see citing works
RNA sequencing: advances, challenges and opportunities
Ozsolak, F., & Milos, P. M. (2011) · Nature Reviews Genetics · doi:10.1038/nrg2934 · Cited by 2,135 · see citing works
“In addition, during first-strand cDNA synthesis, spurious second-strand cDNA artefacts can be introduced, owing to the DNA-dependent DNA polymerase (DDDP) activities of reverse transcriptase”
Background't Hoen, P. A. C., Ariyurek, Y., Thygesen, H. H., Vreugdenhil, E., Vossen, R. H. A. M., de Menezes, R. X., Boer, J. M., van Ommen, G.-J. B., & den Dunnen, J. T. (2008) · Nucleic Acids Research · doi:10.1093/nar/gkn705 · Cited by 742 · see citing works
Preman, P., Moechars, D., Fertan, E., Wolfs, L., Serneels, L., Shah, D., Lamote, J., Poovathingal, S., Snellinx, A., Mancuso, R., et al. (2024) · EMBO Molecular Medicine · doi:10.1038/s44321-024-00162-7 · Cited by 32 · see citing works
- Authors
- Perocchi, F., Xu, Z., Clauder-Münster, S., & Steinmetz, L. M.
- Affiliations
- European Molecular Biology Laboratory (DE)
- Corresponding author
- Steinmetz, Lars M.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2007-09-26
- Volume
- 35
- Issue
- 19
- Pages
- e128
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC2095812
- OpenAlex
- W2107002447
- Cited by
- 193
- References
- 1
- Citations by year
- 2026: 3 · 2025: 2 · 2024: 2 · 2023: 2 · 2022: 2 · 2021: 4 · 2020: 6 · 2019: 3 · 2018: 11 · 2017: 4 · 2016: 7 · 2015: 10 · 2014: 23 · 2013: 13 · 2012: 17
- FWCI
- 6.92
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by-nc
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Funders
- National Human Genome Research Institute (F32HG000205, HG000205, P01HG000205)
- National Institute of General Medical Sciences (R01GM068717, GM 068717)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Keywords
- antisense transcription, actinomycin D, reverse transcription, gene expression profiling, Saccharomyces cerevisiae, tiling arrays, genome-wide transcriptome
- MeSH terms
- Dactinomycin, Saccharomyces cerevisiae, RNA, Antisense, Artifacts, Reverse Transcriptase Polymerase Chain Reaction, Oligonucleotide Array Sequence Analysis, Gene Expression Profiling, Genomics, Reverse Transcription
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