Verbeelen Tom, Van Houdt Rob, Leys Natalie, Ganigué Ramon, Mastroleo Felice
Microbiology Unit, Interdisciplinary Biosciences, Belgian Nuclear Research Centre (SCK CEN), 2400 Mol, Belgium.
Center for Microbial Ecology and Technology (CMET), Faculty of Bioscience Engineering, Ghent University, 9000 Ghent, Belgium.
iScience. 2022 Oct 9;25(11):105311. doi: 10.1016/j.isci.2022.105311. eCollection 2022 Nov 18.
We developed a procedure for extracting maximal amounts of high-quality RNA from low-biomass producing (autotrophic) bacteria for experiments where sample volume is limited. Large amounts of high-quality RNA for downstream analyses cannot be obtained using larger quantities of culture volume. The performance of standard commercial silica-column based kit protocols and these procedures amended by ultrasonication or enzymatic lysis were assessed. The ammonium-oxidizing and nitrite-oxidizing were used as model organisms for optimization of the RNA isolation protocol. Enzymatic lysis through lysozyme digestion generated high-quality, high-yield RNA samples. Subsequent RNA-seq analysis resulted in qualitative data for both strains. The RNA extraction procedure is suitable for experiments with volume and/or biomass limitations, e.g., as encountered during space flight experiments. Furthermore, it will also result in higher RNA yields for whole transcriptome experiments where sample volume and/or biomass was increased to compensate the low-biomass characteristic of autotrophs.
我们开发了一种从低生物量产生（自养）细菌中提取最大量高质量RNA的方法，用于样本量有限的实验。使用大量培养体积无法获得用于下游分析的大量高质量RNA。评估了基于标准商业硅胶柱试剂盒方案的性能以及通过超声处理或酶解修正后的这些方案。将氨氧化菌和亚硝酸盐氧化菌用作优化RNA分离方案的模式生物。通过溶菌酶消化进行酶解产生了高质量、高产量的RNA样本。随后的RNA测序分析为两种菌株提供了定性数据。该RNA提取方法适用于存在体积和/或生物量限制的实验，例如在太空飞行实验中遇到的情况。此外，对于全转录组实验，在增加样本体积和/或生物量以补偿自养生物低生物量特征的情况下，它也将产生更高的RNA产量。
