The guanidine thiocyanate‐high EDTA method for total microbial RNA extraction from severely heavy metal‐contaminated soils

Abstract

Molecular analyses relying on RNA, as a direct way to unravel active microbes and their functional genes, have received increasing attention from environmental researchers recently. However, extracting sufficient and high-quality total microbial RNA from seriously heavy metal-contaminated soils is still a challenge. In this study, the guanidine thiocyanatehigh EDTA (GTHE) method was established and optimized for recovering high quantity and quality of RNA from long-term heavy metal-contaminated soils. Due to the low microbial biomass in the soils, we combined multiple strong denaturants and intense mechanical lysis to break cells for increasing RNA yields. To minimize RNAase and heavy metals interference on RNA integrity, the concentrations of guanidine thiocyanate and EDTA were increased from 0.5 to 0.625 ml g À1 soil and 10 to 100 mM, respectively. This optimized GTHE method was applied to seven severely contaminated soils, and the RNA recovery efficiencies were 2.80~59.41 lg g À1 soil. The total microbial RNA of non-Cr(VI) (NT) and Cr(VI)-treated (CT) samples was utilized for molecular analyses. The result of qRT-PCR demonstrated that the expressions of two tested genes, chrA and yieF, were respectively upregulated 4.12and 62.43-fold after Cr(VI) treatment. The total microbial RNA extracted from NT and CT samples, respectively, reached to 26.70 lg and 30.75 lg, which were much higher than the required amount (5 lg) for metatranscriptomic library construction. Besides, ratios of mRNA read were more than 86%, which indicated the high-quality libraries constructed for metatranscriptomic analysis. In summary, the GTHE method is useful to study microbes of contaminated habitats.

Comparative Metagenomic and Metatranscriptomic Analyses Reveal the Response of Black Soldier Fly (Hermetia illucens) Larvae Intestinal Microbes and Reduction Mechanisms to High Concentrations of Tetracycline

“…DNA and RNA Extraction, Library Construction, Sequencing E.Z.N.A Soil Kit (Omega Bio-Tek, Norcross, GA) was used to extract gDNA from BSFL intestinal microbiota following the manufacturer's instructions. The RNA of BSFL intestinal microbiota were extracted using our previously developed thiocyanate-high EDTA method [46]. A NanoDrop ND-2000 spectrophotometer (Thermo Fisher, MA, USA) and agarose gel electrophoresis were first used to determine the quantity and quality of gDNA/RNA.…”

Soil metatranscriptomics: An improved RNA extraction method toward functional analysis using nanopore direct RNA sequencing

“…Therefore, the development of efficient high-quality RNA extraction protocols for different soil sample types is required. To extract RNA from a variety of soil types, different manual extraction methods have been optimized (Lever et al 2015; Lim et al 2016; Mettel et al 2010; Paulin et al 2013; Pei et al 2021; Peršoh et al 2008; Qin et al 2016; Sharma et al 2012; Thorn et al 2019; Wang et al 2012b). However, soil samples exhibit many heterogeneities that have hampered the development of a universal RNA extraction method.…”