Deep sequencing reveals complex spurious transcription from transiently transfected plasmids

PLoS One. 2012;7(8):e43283. doi: 10.1371/journal.pone.0043283. Epub 2012 Aug 16.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Flow Cytometry
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Plasmids / genetics*
  • RNA, Antisense / genetics

Substances

  • RNA, Antisense

Grant support

This research was supported by the Czech Science Foundation (grant GACR 204/09/0085), institutional support AV0Z50520514 and RVO 68378050, and the European Molecular Biology Organization Strategic Development Installation Grant (program 0001488, partial). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.