title: A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil.
abstract: The lack of a universal method to extract RNA from soil hinders the progress of studies related to nitrification in soil, which is an important step in the nitrogen cycle. It is particularly difficult to extract RNA from certain types of soils such as Andosols (volcanic ash soils), which is the dominant agricultural soil in Japan, because of RNA adsorption by soil. To obtain RNA from these challenging soils to study the bacteria involved in nitrification, we developed a soil RNA extraction method for gene expression analysis. Autoclaved casein was added to an RNA extraction buffer to recover RNA from soil, and high-quality RNA was successfully extracted from eight types of agricultural soils that were significantly different in their physicochemical characteristics. To detect bacterial ammonia monooxygenase subunit A gene (amoA) transcripts, bacterial genomic DNA and messenger RNA were co-extracted from two different types of Andosols during incubation with ammonium sulfate. Polymerase chain reaction-denaturing gradient gel electrophoresis and reverse transcription polymerase chain reaction-denaturing gradient gel electrophoresis analyses of amoA in soil microcosms revealed that only few amoA, which had the highest similarities to those in Nitrosospira multiformis, were expressed in these soils after treatment with ammonium sulfate, although multiple amoA genes were present in the soil microcosms examined.
authors.affiliation: Environmental Biofunction Division, National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki, 305-8604, Japan.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.forename: Kazunari
authors.initials: K
authors.lastname: Nagaoka
authors.forename: Masahito
authors.initials: M
authors.lastname: Hayatsu
authors.forename: Yoriko
authors.initials: Y
authors.lastname: Sakai
authors.forename: Kanako
authors.initials: K
authors.lastname: Tago
authors.forename: Susumu
authors.initials: S
authors.lastname: Asakawa
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fujii
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D007564:Japan; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D043369:Nitrosomonadaceae / Q000201:enzymology / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA; D012987:Soil / Q000737:chemistry*; D012988:Soil Microbiology; D018707:Volcanic Eruptions
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D004269:DNA, Bacterial; D012987:Soil; D012313:RNA; D010088:Oxidoreductases; C045582:ammonia monooxygenase