title: Isolation of high-quality total RNA from rumen anaerobic bacteria and fungi, and subsequent detection of glycoside hydrolases.
abstract: The rumen is one of the most powerful fibrolytic fermentation systems known. Gene expression analyses, such as reverse transcription PCR (RT-PCR), microarrays, and metatranscriptomics, are techniques that could significantly expand our understanding of this ecosystem. The ability to isolate and stabilize representative RNA samples is critical to obtaining reliable results with these procedures. In this study, we successfully isolated high-quality total RNA from the solid phase of ruminal contents by using an improved RNA extraction method. This method is based on liquid nitrogen grinding of whole ruminal solids without microbial detachment and acid guanidinium - phenol - chloroform extraction combined with column purification. Yields of total RNA were as high as 150 µg per g of fresh ruminal content. The typical large subunit/small subunit rRNA ratio ranged from 1.8 to 2.0 with an RNA integrity number (Agilent Technologies) greater than 8.5. By eliminating the detachment step, the resulting RNA was more representative of the complete ecosystem. Our improved method removed a major barrier limiting analysis of rumen microbial function from a gene expression perspective. The polyA-tailed eukaryotic mRNAs obtained have successfully been applied to next-generation sequencing, and metatranscriptomic analysis of the solid fraction of rumen contents revealed abundant sequences related to rumen fungi.
authors.affiliation: Agriculture and Agri-Food Canada, Lethbridge Research Centre.
authors.forename: Pan
authors.initials: P
authors.lastname: Wang
authors.forename: Meng
authors.initials: M
authors.lastname: Qi
authors.forename: Perry
authors.initials: P
authors.lastname: Barboza
authors.forename: Mary Beth
authors.initials: MB
authors.lastname: Leigh
authors.forename: Emilio
authors.initials: E
authors.lastname: Ungerfeld
authors.forename: L Brent
authors.initials: LB
authors.lastname: Selinger
authors.forename: Tim A
authors.initials: TA
authors.lastname: McAllister
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Forster
mesh_terms: D000818:Animals; D001421:Bacteria, Anaerobic / Q000235:genetics / Q000302:isolation & purification*; D017753:Ecosystem; D005658:Fungi / Q000235:genetics / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000235:genetics*; D012329:RNA, Bacterial / Q000302:isolation & purification*; D012331:RNA, Fungal / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012417:Rumen / Q000382:microbiology*
chemical_list: D012329:RNA, Bacterial; D012331:RNA, Fungal; D006026:Glycoside Hydrolases