Rapid Method for Coextraction of DNA and RNA from Natural Environments for Analysis of Ribosomal DNA- and rRNA-Based Microbial Community Composition

Abstract

A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.The molecular analysis of 16S rRNA is now central to studies examining the diversity of microorganisms in the environment. Traditional methods based upon cultivation underestimate diversity considerably, whereas modern molecular methods (PCR, cloning, and sequencing) have provided a greater insight into the extent of prokaryotic diversity (for a review see reference 6). Methodologies for the analyses of a DNA-based phylogeny (using the 16S rRNA gene) are now well established but the direct targeting of 16S rRNA, as a potential indicator of activity (4), has received comparatively less attention, due primarily to the lack of suitable protocols for extraction from natural environments.Methods currently employed for DNA extraction vary widely, from direct methods of in situ lysis to indirect methods of initial cell extraction prior to lysis. In both cases, the methods used often include various combinations of bead beating, detergents, enzymatic lysis, and solvent extractions to obtain a crude preparation of nucleic acid (see, e.g., references 5 and 8). The utility of the published methods varies, particularly in soil systems, since inhibitory compounds such as humic acids and clay minerals are often coextracted. Therefore, additional purification procedures are required for successful PCR amplification. These additional steps can prevent the simultaneous extraction of the labile RNA (3) and reduce DNA yield. Reliable extraction methods have been reported for isolation of RNA from soils (2, 3, 11) and other environments (10), but they typically involve multiple steps for purification, rendering them impractical for processing large numbers of samples. Here we describe the first direct method for the rapid coextraction of RNA and DNA from soil for the comparison of bacterial diversity by 16S rRNA reverse transcription-PCR (RT-PCR) and 16S ribosomal DNA (rDNA)-PCR. To demonstrate the efficacy and reproducibility of the method, we present the denaturing gradient gel electrophoresis (DGGE) analysis of the diversity of bacterial populations in a humified upland soil based on 16S rDNA and 16S rRNA templates.Sampling and extraction protocol. Replicate cores of a brown forest soil (pH 4.5 to 5.0) were collected from the Sourhope Field Experiment Site in the Scottish Borders (United Kingdom) to a depth of 20 to 25 cm. Each replicate core was divided into four horizons characterized by standard nomenclature (Fh, H, Ah upper, and Ah lower). Prior to nucleic acid extraction, all solutions and glassware were rendered RNase free by diethyl pyrocarbonate...

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