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Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcr...

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Autores principales: Nejepinska, Jana, Malik, Radek, Moravec, Martin, Svoboda, Petr
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420890/
https://www.ncbi.nlm.nih.gov/pubmed/22916237
http://dx.doi.org/10.1371/journal.pone.0043283
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author Nejepinska, Jana
Malik, Radek
Moravec, Martin
Svoboda, Petr
author_facet Nejepinska, Jana
Malik, Radek
Moravec, Martin
Svoboda, Petr
author_sort Nejepinska, Jana
collection PubMed
description Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcription may have undesirable effects as some plasmids, when co-transfected, inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to a Kan/Neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression from transiently transfected plasmids underscores the importance of appropriate experimental controls.
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spelling pubmed-34208902012-08-22 Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids Nejepinska, Jana Malik, Radek Moravec, Martin Svoboda, Petr PLoS One Research Article Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcription may have undesirable effects as some plasmids, when co-transfected, inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to a Kan/Neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression from transiently transfected plasmids underscores the importance of appropriate experimental controls. Public Library of Science 2012-08-16 /pmc/articles/PMC3420890/ /pubmed/22916237 http://dx.doi.org/10.1371/journal.pone.0043283 Text en © 2012 Nejepinska et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Nejepinska, Jana
Malik, Radek
Moravec, Martin
Svoboda, Petr
Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids
title Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids
title_full Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids
title_fullStr Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids
title_full_unstemmed Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids
title_short Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids
title_sort deep sequencing reveals complex spurious transcription from transiently transfected plasmids
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420890/
https://www.ncbi.nlm.nih.gov/pubmed/22916237
http://dx.doi.org/10.1371/journal.pone.0043283
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