Isolation of RNA from bacterial samples of the human gastrointestinal tract

Abstract

The human gastrointestinal (GI) tract contains a complex microbial community that consists of numerous uncultured microbes. Therefore, nucleic-acid-based approaches have been introduced to study microbial diversity and activity, and these depend on the proper isolation of DNA, rRNA and mRNA. Here, we present an RNA isolation protocol that is suitable for a wide variety of GI tract samples. The procedure for isolating DNA from GI tract samples is described in another Nature Protocols article. One of the benefits of our RNA isolation protocol is that sampling can be performed outside the laboratory, which offers possibilities for implementation in large intervention studies. The RNA isolation is based on mechanical disruption, followed by isolation of nucleic acids using phenol:chloroform:isoamylalcohol extraction and removal of DNA. In our laboratory, this protocol has resulted in the isolation of rRNA and mRNA of sufficient quality and quantity for microbial diversity and activity studies. Depending on the number of samples, the sample type and the quenching procedure chosen, the whole procedure can be performed within 2.5-4 h.

Metaproteomics Approach To Study the Functionality of the Microbiota in the Human Infant Gastrointestinal Tract

“…Microbial cells were released from the feces and washed as previously described (5,22). The bead beating method was confirmed to be applicable for protein extraction of infant fecal microbiota containing bifidobacteria (data not shown), as expected from previous systematic studies (5,14,20,23,24). Total soluble protein was obtained by three treatments of 45 s of bead beating (FastPrep; Qbiogene), interspersed by 1 min on ice, in 500-l isoelectric focusing (IEF) buffer (Fluka, Switzerland) (10 M urea) containing 2% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) (Roche, Switzerland), 0.65 mM dithiothreitol (DTT) (Sigma, Switzerland), 0.2% Biolytes 3/10 (Bio-Rad), Pefabloc Sc (Fluka), and glass beads (Sigma) (Յ0.1 mm).…”

Comparative Analysis of Lactobacillus plantarum WCFS1 Transcriptomes by Using DNA Microarray and Next-Generation Sequencing Technologies

“…Total RNA was extracted from the cell pellets according to the Macaloid-based RNA isolation protocol (52). Extraction was followed by RNA purification using the RNAeasy minikit (Qiagen), including an on-column DNase I (Roche, Germany) treatment as described previously (52). The enrichment of mRNA was performed by the selective removal of 16S and 23S rRNA using oligonucleotide probes attached to magnetic beads according to the manufacturer's protocol (Microbexpress; Ambion, Applied Biosystems, Niewerkerk a/d Ijssel, the Netherlands) (44).…”

Mixed-Species Genomic Microarray Analysis of Fecal Samples Reveals Differential Transcriptional Responses of Bifidobacteria in Breast- and Formula-Fed Infants

“…qPCR analysis of DNA extracts was performed in triplicate as described previously (20) Total RNA isolation. Fecal samples were collected at three time points and immediately mixed with RNAlater (Ambion Inc., Austin, TX) at a ratio of 1:2 (wt/vol), and the mixtures were incubated overnight at 4°C and stored at Ϫ20°C until further processing as described previously (70).…”