An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene

PLoS One. 2021 Nov 15;16(11):e0260002. doi: 10.1371/journal.pone.0260002. eCollection 2021.

Abstract

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Culture Media / chemistry*
  • Phenol / chemistry
  • Phenylenediamines / chemistry
  • Polyurethanes / chemistry*
  • Pseudomonas / genetics
  • Pseudomonas / growth & development*
  • RNA, Bacterial / isolation & purification*
  • RNA, Bacterial / standards
  • Xenobiotics / chemistry

Substances

  • Culture Media
  • Phenylenediamines
  • Polyurethanes
  • RNA, Bacterial
  • Xenobiotics
  • Phenol
  • 2,4-diaminotoluene

Supplementary concepts

  • Pseudomonas capeferrum

Grants and funding

The Financial support by the European Union’s Horizon 2020 Research and Innovation Program under grant agreement no. 633962 for the project P4SB is greatly appreciated. Additionally, this work was supported by the German Federal Ministry of Education and Research via the project NO STRESS under grant number 031B085C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.