RNA isolation from soil for bacterial community and functional analysis: evaluation of different extraction and soil conservation protocols

• Published in: Journal of microbiological methods 2002-10, Vol.51 (2), p.171-179
• Main Authors: Sessitsch, Angela, Gyamfi, Stephen, Stralis-Pavese, Nancy, Weilharter, Alexandra, Pfeifer, Ulrike
• Format: Article
• Language: English
• Subjects: 16S rRNA; Glutamate-Ammonia Ligase - genetics; Polymorphism, Restriction Fragment Length; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Bacterial - isolation & purification; RNA, Ribosomal, 16S - genetics; Soil; Soil Microbiology; T-RFLP analysis
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(02)00065-9

The impact of three different RNA isolation methods on the community analysis of metabolically active bacteria was determined by reverse transcription (RT) and PCR amplification of 16S rRNA genes and subsequent terminal restriction fragment length polymorphism (T-RFLP) analysis. Furthermore, soil samples were stored at different conditions in order to evaluate the effect of soil conservation methods on the outcome of the population analysis. The quality of mRNA was assessed by reverse transcription and PCR amplification of eubacterial glutamine synthetase genes. Our results indicated that the community composition as well as the abundance of individual members were affected by the kind of RNA isolation method. Furthermore, the extraction method influenced the recovery of mRNA. Lyophilization, storage at −20 °C as well as storage in glycerol stocks at −80 °C proved to be equally appropriate for the storage of soils and subsequent RNA isolation.