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<p>RNA-based assays hold great potential for assessing viability molecularly in slow- or non-growing mycobacteria, and RNAseq has evolved into a powerful tool in infectious disease research. Such applications require efficient RNA preservation for optimal results. The performance of 70% ethanol as simpler alternative to commonly-used GTC-based storage of mycobacteria at − 80&#xa0;°C was compared on cultured <i>Mycobacterium tuberculosis</i> H37Ra subjected to following setups: a 45&#xa0;°C heat shock to test immediate stabilisation, five freeze-thaw scenarios to mimic different shipping conditions, and long-term storage at -80&#xa0;°C, -20&#xa0;°C, 4&#xa0;°C or 30&#xa0;°C for up to twelve months. Treatment with 70% ethanol yielded overall higher RNA quantities compared to GTC-TCEP and RNA integrity was maintained at -20&#xa0;°C for twelve months. Both buffers are reportedly mycobactericidal and, in this study, prevented heat stress-induced transcriptomic changes, thereby conserving a transcriptomic snapshot. RNA yield and integrity remained unaltered after treatment with 70% ethanol, even with up to three freeze-thaw cycles. Based on these results, we recommend 70% ethanol over GTC-TCEP for RNA preservation of mycobacterial cultures. While freeze-thawing, short-term high-temperature and − 20&#xa0;°C long-term storage results are promising, this inexpensive, widely available buffer needs further validation prior to applying it for RNA-based analysis in clinical samples.</p>

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70% ethanol preserves mycobacterial RNA from cultures more efficiently than GTC-TCEP

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年4月10日

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  • Lena Krausser,
  • Magalie Van Dyck-Lippens,
  • Ramata Balde,
  • Rabab Reenaers,
  • Reem Al Mubarak,
  • Leen Rigouts,
  • Nicholas D. Walter,
  • Martin I. Voskuil,
  • Annelies Van Rie,
  • Bouke C. de Jong,
  • Sofie M. Braet

摘要

RNA-based assays hold great potential for assessing viability molecularly in slow- or non-growing mycobacteria, and RNAseq has evolved into a powerful tool in infectious disease research. Such applications require efficient RNA preservation for optimal results. The performance of 70% ethanol as simpler alternative to commonly-used GTC-based storage of mycobacteria at − 80 °C was compared on cultured Mycobacterium tuberculosis H37Ra subjected to following setups: a 45 °C heat shock to test immediate stabilisation, five freeze-thaw scenarios to mimic different shipping conditions, and long-term storage at -80 °C, -20 °C, 4 °C or 30 °C for up to twelve months. Treatment with 70% ethanol yielded overall higher RNA quantities compared to GTC-TCEP and RNA integrity was maintained at -20 °C for twelve months. Both buffers are reportedly mycobactericidal and, in this study, prevented heat stress-induced transcriptomic changes, thereby conserving a transcriptomic snapshot. RNA yield and integrity remained unaltered after treatment with 70% ethanol, even with up to three freeze-thaw cycles. Based on these results, we recommend 70% ethanol over GTC-TCEP for RNA preservation of mycobacterial cultures. While freeze-thawing, short-term high-temperature and − 20 °C long-term storage results are promising, this inexpensive, widely available buffer needs further validation prior to applying it for RNA-based analysis in clinical samples.