Extraction of bacterial RNA from soil: challenges and solutions

Abstract

Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them.

Fig. 1

The behavior of humic and fulvic acids during phenol extraction and ethanol precipitation. Humic and fulvic acids were prepared as previously described (139). Citrate-saturated phenol at pH 4.3 was used for extraction, and water was used as a control to show the original color of the phenol reagent used. The aqueous layer was transferred to a fresh tube, followed by the addition of 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol for precipitation.

Fig. 2

Ultraviolet-visible absorption spectra of RNA samples (A) and the magnified spectra by adjustment of scale (B). Marker-H and Marker-L, Novagen Perfect RNA Marker (0.2–10 Kb) at high (H) or low (L) concentrations; KT2440-H and KT2440-L, a highly purified RNA sample prepared from a pure culture of Pseudomonas putida KT2440 strain at high (H) or low (L) concentrations; Soil-1 and Soil-2, two humic-contaminated RNA samples prepared from soil. Vertical lines indicate the positions of absorbance at 320 nm and 400 nm, respectively.