A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil
A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil
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DOI：
10.1007/s00253-012-4342-3
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发表时间：
2012-08
影响因子：
5
通讯作者：
Yong Wang;K. Nagaoka;M. Hayatsu;Yoriko Sakai;Kanako Tago;S. Asakawa;T. Fujii
中科院分区：
文献类型：
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作者：
Yong Wang;K. Nagaoka;M. Hayatsu;Yoriko Sakai;Kanako Tago;S. Asakawa;T. Fujii
缺乏从土壤中提取RNA的通用方法阻碍了与土壤硝化作用相关的研究进展，而土壤硝化作用是氮循环中的一个重要步骤。由于土壤对核糖核酸的吸附，从某些类型的土壤中提取核糖核酸尤其困难，例如安多索(火山灰土壤)，这是日本的主要农业土壤。为了从这些具有挑战性的土壤中提取RNA来研究参与硝化作用的细菌，我们开发了一种用于基因表达分析的土壤RNA提取方法。将高压灭菌的酪蛋白加入到RNA提取缓冲液中，从土壤中回收RNA，成功地从8种理化性质明显不同的农业土壤中提取出高质量的RNA。为了检测细菌氨单加氧酶A亚单位(AmoA)基因的转录本，从两种不同类型的安多沙土中提取细菌基因组DNA和信使RNA。聚合酶链式反应-变性梯度凝胶电泳法和逆转录-聚合酶链式反应-变性梯度凝胶电泳法分析表明，土壤微宇宙经硫酸铵处理后，虽然存在多个amoA基因，但只有与多形硝酸螺旋体相似性最高的少数几个amoA基因在土壤微宇宙中表达。
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 ofamoAin soil microcosms revealed that only fewamoA, which had the highest similarities to those inNitrosospira multiformis, were expressed in these soils after treatment with ammonium sulfate, although multipleamoAgenes were present in the soil microcosms examined.