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.

Expression of denitrification genes in response to a waterlogging event in a Fluvisol and its relationship with large nitrous oxide pulses

“…Various types of soil have different characteristics in terms of RNA adsorption (Wang et al ., ). Thus, in a preliminary experiment, we performed RNA extraction from soil using a lysis buffer containing different quantities of casein to investigate the optimal concentration of casein in the lysis buffer as described in a previous report (Wang et al ., ). We found that RNA adsorption by a Fluvisol, which was used in the current experiment, could be ignored so that casein was not necessary for RNA extraction from this soil.…”

“…We found that RNA adsorption by a Fluvisol, which was used in the current experiment, could be ignored so that casein was not necessary for RNA extraction from this soil. To facilitate RNA extraction from the small amount of soil, RNA was extracted from the RNAlater ® ‐ICE‐treated soil using a ZR Soil/Fecal RNA MicroPrep™ kit (Zymo Research) with modifications, according to a published method (Wang et al ., , ). Briefly, 0.4 g of soil amended with 0.8 mL Na 2 HPO 4 /NaH 2 PO 4 buffer (300 mM, pH 7) was subjected to bead beating at 5.5 m s −1 for 30 s in a FastPrep ® FP120 machine (Qbiogene).…”

“…Because the quality (integrity and purity) of RNA is crucial for RNA detection, extraction of RNA from soil has been studied for a long time, and there are many approaches to extract RNA from soil. In the current study, to obtain high‐quality RNA for qRT‐PCR detection, RNA extraction from soil was performed with a method (Wang et al ., , ), which has been proved to acquire high‐quality RNA from soil for detection of mRNA (Wang et al ., , ; Iino et al ., ; Yoshida et al ., ).…”

Organic soil amendment with Spent Mushroom Substrate results in fungal colonisation, alters bacterial-fungal co-occurrence patterns and improves plant productivity

“…DNA and RNA were co-extracted from 0.5 g of soil using the method described by Griffiths et al (2000), modified with the addition of casein to increase the nucleic acid yield, as demonstrated by Wang et al (2012). To remove remaining PCR inhibitors, the OneStep PCR inhibitor removal kit (Zymo Research, Irvine, CA, USA) was employed.…”

Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

“…In environmental microbiology it is an invaluable approach to further link microbial activity, via gene expression, to microbial and ecosystem processes, compared to DNA approaches alone (Smith and Osborn, 2009;Saleh-Lakha et al, 2011;Gadkar and Filion, 2013). As a result the approach has been used to quantify transcripts to distinguish different pathways of the nitrogen cycle in sediments (Santoro et al, 2010;Smith et al, 2007;Zheng et al, 2013;Damashek et al, 2015;Duff et al, 2017;Santos et al, 2018;Zhang et al, 2018), soil (Leininger et al, 2006;Graham et al, 2011;Wang et al, 2012;Li et al, 2017;Pierre et al, 2017), water column (Tolar et al, 2016;Santoro et al, 2010;Kapoor et al, 2015;Posman et al, 2017;Feng et al, 2018;Gonçalves et al, 2018;Liu et al, 2018;Christiansen et al, 2019) and other microbial processes including water treatment (Gadkar and Filion, 2013;Botes et al, 2013;Wang et al, 2016;Pelissari et al, 2017Pelissari et al, , 2018 and bioremediation (Gadkar and Filion, 2013;Marzorati et al, 2010;Yergeau et al, 2009). In addition to this, cDNA from mRNA or rRNA can undergo PCR for amplicon sequencing to reveal actively transcribing organisms within the environment (Cholet et al, 2019;Zhang et al, 2018;Duff et al, 2017).…”