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A modified approach for high-quality RNA extraction of spore-forming Bacillus subtilis at varied physiological stages

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Abstract

Background

High quality RNA is required for the molecular study. Sample preparation of the spore-forming, Gram-positive bacteria like Bacillus sp., remains challenging although several methods have been proposed. Those techniques were simply developed using cell samples at certain growth stages despite some molecular studies like transcriptomic analyses require RNA samples from different physiological stages.

Methods and results

We developed the rapid, simple yet effective cell-lysis technique with limit use of harsh reagents by modifying the kit-based protocols. Appropriate lysozyme loading (20 mg/mL), incubation time (30 min), and temperature (37 °C) enabled cell lysis and enhanced RNA extraction from both vegetative cells and endospores of Bacillus subtilis TL7-3. High RNA Integrity Numbers and ratios of A260/A280 and A260/A230 of all RNA products collected during the batch cultivation confirmed that invert mixing with absolute ethanol prevented RNA damage during protein denaturation. With the process modification of the major steps in cell lysis and RNA extraction compared with the kit-based protocols that are typically used in laboratory work, interestingly, our modified protocol, simple-yet-effective, yielded higher concentration, purity, and integrity of RNA products from all cell samples collected at different physiological stages. While the kit-based protocols either failed to provide high RNA concentration or RNA purity and integrity for all cell samples particularly during the late-log, stationary, or sporulation.

Conclusions

Therefore, we can claim the significance of this modified protocol to be applicable for RNA extraction to those spore-forming Gram-positive bacteria not limited to B. subtilis growing at varied physiological stages.

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Data availability

The 16S rRNA gene sequence of B. subtilis TL7-3 has been submitted to NCBI with accession number MW820294.

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Acknowledgements

P. Jaiaue is the recipient of the Development and Promotion of Science and Technology Talents Project (DPST) provided by Thailand Research Fund. The authors would like to acknowledge National Security and Dual-Use Technology Center, National Science and Technology Development Agency (NSTDA), for supporting our study.

Funding

This study was partially funded by National Research Council of Thailand; Research Chair Professor Grant provided by National Science and Technology Development Agency (NSTDA); and Thailand Research Fund [Grant No. RTA6280014].

Author information

Authors and Affiliations

  1. Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand

    Phetcharat Jaiaue

  2. Research Unit in Bioconversion and Bioseparation for Value-Added Chemical Production, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand

    Piroonporn Srimongkol, Sitanan Thitiprasert & Nuttha Thongchul

  3. Research Unit in Bioconversion and Bioseparation for Value-Added Chemical Production, Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand

    Somboon Tanasupawat

  4. Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkla, Thailand

    Benjamas Cheirsilp

  5. Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand

    Suttichai Assabumrungrat

Authors
  1. Phetcharat Jaiaue
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  2. Piroonporn Srimongkol
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  7. Nuttha Thongchul
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Contributions

PJ performed the experiments and statistical analysis. PS, ST, and NT conceived the research idea, designed the methodology, and scrutinized the experimental data. ST provided the strain in this study and visualized the experimental data. BC, SA, and NT provided research funding and supervised the research work. PJ and NT wrote and edited the manuscript.

Corresponding authors

Correspondence to Sitanan Thitiprasert or Nuttha Thongchul.

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Jaiaue, P., Srimongkol, P., Thitiprasert, S. et al. A modified approach for high-quality RNA extraction of spore-forming Bacillus subtilis at varied physiological stages. Mol Biol Rep 48, 6757–6768 (2021). https://doi.org/10.1007/s11033-021-06673-7

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  • DOI: https://doi.org/10.1007/s11033-021-06673-7

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