An extraction method was developed to recover high-quality RNA from rumen 
digesta and mouse feces for phylogenetic analysis of metabolically active 
members of the gut microbial community. Four extraction methods were tested on 
different amounts of the same samples and compared for efficiency of recovery 
and purity of RNA. Trizol extraction after bead beating produced a higher 
quantity and quality of RNA than a similar method using phenol/chloroform. 
Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery 
compared with phosphate-buffered saline during the dissociation of 
microorganisms from rumen digesta or fecal particles. The identity of 
metabolically active bacteria in the samples was analyzed by sequencing 87 
amplicons produced using bacteria-specific 16S rDNA primers, with cDNA 
synthesized from the extracted RNA as the template. Amplicons representing the 
major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; 
Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, 
showing that development of the RNA extraction method enables RNA-based analysis 
of metabolically active bacterial groups from the rumen and other environments. 
Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA 
amplicons were related to the Proteobacteria, providing the first evidence that 
this group may have greater importance in rumen metabolism than previously 
attributed by DNA-based analysis.