Assessment of rhizosphere microbial activity using optimized RNA extraction coupled with universal ribosomal RNA depletion techniques

Metatranscriptomics, which analyzes gene expression patterns in a heterogeneous community, is a powerful tool to evaluate microbial functional activity. A key challenge in this process is obtaining high-quality RNA, which is complicated by the composition and ecosystem complexity of the soil matrix. Another crucial step involves the removal of highly abundant ribosomal RNA (rRNA), as its presence can dominate sequencing results and obscure the detection of messenger RNA (mRNA) expression. Conventional library preparation methods often struggle to efficiently remove rRNA from a complex mix of prokaryotic and eukaryotic organisms, further complicating mRNA isolation. To overcome these limitations, we have developed an optimized method for extracting RNA from soybean rhizosphere microbes, followed by universal rRNA depletion to create rRNA-free samples for sequencing. These samples were sequenced using an Illumina high-throughput sequencer. Our optimized cetyltrimethylammonium bromide (CTAB) phenol–chloroform extraction protocol significantly improved RNA yield and quality from clay-rich soils, outperforming previously published methods and commercial kits. Illumina sequencing data revealed minimal rRNA contamination, and the resulting short reads could be successfully assembled into transcripts. These findings also demonstrate that using the Zymo-Seq RiboFree Total RNA Library Kit effectively enabled library preparation from complex environmental samples for Illumina sequencing. This RNA sample preparation method, combined with our optimized extraction technique, provides a valuable approach for studying rhizosphere microbes that hold exciting potential for advancing soil health assessments and understanding plant–microbe-pathogen interactions.