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.

中文翻译：

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不同生理阶段产芽孢枯草芽孢杆菌高质量 RNA 提取的改进方法

背景

分子研究需要高质量的 RNA。尽管已经提出了几种方法，但对芽孢杆菌等革兰氏阳性细菌的样品制备仍然具有挑战性。尽管一些分子研究（如转录组分析）需要来自不同生理阶段的 RNA 样本，但这些技术只是使用特定生长阶段的细胞样本开发的。

方法和结果

我们通过修改基于试剂盒的方案，开发了快速、简单但有效的细胞裂解技术，限制了苛刻试剂的使用。适当的溶菌酶负载 (20 mg/mL)、孵育时间 (30 分钟) 和温度 (37 °C) 能够使细胞裂解并增强从枯草芽孢杆菌TL7-3 的营养细胞和内生孢子中提取的 RNA。_{A 260} /A ₂₈₀和 A ₂₆₀ /A ₂₃₀的高 RNA 完整性数量和比率分批培养期间收集的所有 RNA 产物证实，与无水乙醇反向混合可防止蛋白质变性过程中的 RNA 损伤。与实验室工作中通常使用的基于试剂盒的方案相比，细胞裂解和 RNA 提取中主要步骤的工艺修改，有趣的是，我们修改后的方案简单而有效，产生了更高的浓度、纯度和完整性在不同生理阶段收集的所有细胞样本的 RNA 产物。虽然基于试剂盒的方案无法为所有细胞样品提供高 RNA 浓度或 RNA 纯度和完整性，特别是在后期、静止或孢子形成期间。

结论

因此，我们可以声称这一修改后的方案的意义在于适用于那些形成孢子的革兰氏阳性细菌的 RNA 提取，而不仅限于在不同生理阶段生长的枯草芽孢杆菌。