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.

FIG. 1

(a) Negative image of a 1% ethidium bromide-stained agarose gel of the total nucleic extract. Lane 1, HyperLadder I (Bioline, London, United Kingdom); lane 2, total nucleic extract from 0.5 g of Sourhope soil. (b) Ethidium bromide (1.5%)-stained agarose gel showing PCR and RT-PCR amplification products from each soil horizon (Fh, H, Ah upper [AhU], and Ah lower [AhL]). Lane 1, HyperLadder I; lanes 2 to 5 amplification products from 16S rDNA for each soil horizon; lanes 6 to 9, amplification products from reverse-transcribed 16S rRNA; lanes 10 to 13, amplified 16S rRNA (controls without reverse transcriptase) for each horizon; lane 14, amplified bacterial 16S rDNA (positive control); lane 15, no-template negative control.

FIG. 2

Scanned image of a silver-stained DGGE gel (10% acrylamide, 30 to 60% denaturant) profiling the microbial communities by soil horizon and nucleic acid template. For each horizon, two replicate profiles from two independently extracted soil cores are displayed.

FIG. 3

Dendrogram showing clustering analyses of the digitized profiles from Fig. 2, using the unweighted pairwise grouping method with mathematical averages (Dice coefficient of similarity). The analyses takes into account the presence or absence of bands at certain positions in each lane, standardized across the gel using R_f values.