Direct ribosome isolation from soil to extract bacterial rRNA for community analysis.
A simple method that combines an adapted ribosome isolation method and a common RNA extraction step has been developed for selective recovery of intact rRNA from natural microbial communities in soil. After mechanical cell lysis, ribosomes are separated by centrifugation steps, avoiding massive humic acid contamination and RNA degradation. The protocol accommodates the complex composition of soils by blocking adsorbing surfaces and humic acids with polyvinylpyrrolidone and bovine serum albumin. A usual RNA extraction step yields rRNA accessible for hybridization or reverse transcription-PCR. Hybridization with specific oligonucleotide probes was used for group-specific yield comparison. By using a probe hybridizing to the 16S rRNA of the bacterial kingdom, total bacterial rRNA yield was estimated to be in the range of 0.2 microgram per g for different soils. Group-specific probes did not indicate a selectivity of the isolation procedure and differentiated the compositions of different soil microbial communities. The sequence diversity of the isolated RNA population was also revealed by temperature gradient gel electrophoresis of reverse transcription-PCR amplification products by using a region of the 16S rRNA as a target. The pattern obtained by this analysis differed from a similar one resulting from the separation of amplification products of community DNA preparations. This different view of the community composition is attributable to the correlation of ribosome numbers to the metabolic activity of bacteria in the habitat under observation.
Felske
A
A
Biologische Bundesanstalt für Land-und Forstwirtschaft, Institut für Biochemie und Pflanzenvirologie, Braunschweig, Germany.
Engelen
B
B
Nübel
U
U
Backhaus
H
H
D001431:Bacteriological Techniques; D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D017753:Ecosystem; D005069:Evaluation Studies as Topic; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification*; D012270:Ribosomes / Q000737:chemistry; D012988:Soil Microbiology
D015342:DNA Probes; D012329:RNA, Bacterial; D012335:RNA, Ribosomal