Optimization of RNA extraction for bacterial whole transcriptome studies of low-biomass samples

iScience. 2022 Oct 9;25(11):105311. doi: 10.1016/j.isci.2022.105311. eCollection 2022 Nov 18.

ABSTRACT

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 Nitrosomonas europaea and nitrite-oxidizing Nitrobacter winogradskyi 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.

PMID:36345334 | PMC:PMC9636042 | DOI:10.1016/j.isci.2022.105311

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