Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils.
We developed an improved protocol, allowing the simultaneous extraction of DNA and RNA from soil using phenol-chloroform with subsequent column-based separation of DNA and RNA (PCS). We compared this new approach with the well established protocol published by Griffiths et al. (2000), where DNA and RNA are separated by selective enzymatic digestions and two commercial kits used for DNA or RNA extraction, respectively, using four different agricultural soils. We compared yield and purity of the nucleic acids as well as abundance and diversity profiles of the soil bacterial communities targeting the nosZ gene via quantitative real-time PCR and terminal restriction fragment length polymorphism on DNA and RNA level. The newly developed protocol provided purer nucleic acid extracts compared to the used kit-based protocols. All protocols were suitable for DNA- and RNA-based gene quantification, however high variations between replicates were obtained for RNA samples using the original Griffiths protocol. Diversity patterns of nosZ were highly influenced by the extraction protocol used both on the DNA and RNA level. Finally, our data showed that the new protocol allows a simultaneous and reproducible extraction and separation of DNA and RNA, which were suitable for reliable analyses of gene and transcript copy numbers and diversity pattern.
Technische Universität München, Chair of Soil Ecology, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. stefanie.toewe@helmholtz-muenchen.de
Stefanie
S
Töwe
Stefanie
S
Wallisch
Andrea
A
Bannert
Doreen
D
Fischer
Brigitte
B
Hai
Felix
F
Haesler
Kristina
K
Kleineidam
Michael
M
Schloter
D001419:Bacteria / Q000145:classification / Q000235:genetics; D044822:Biodiversity; D002845:Chromatography / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D054892:Metagenome; D012313:RNA / Q000302:isolation & purification*; D012988:Soil Microbiology
D012313:RNA; D004247:DNA