Extraction of Bacterial RNA from Soil: Challenges and Solutions

Wang, Yong; Hayatsu, Masahito; Fujii, Takeshi

doi:10.1264/jsme2.me11304

Cited by 58 publications

(39 citation statements)

References 151 publications

(245 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Absence of genomic DNA was confirmed by quantitative PCR without reverse transcriptase. Successful extraction of total RNA from micro-organisms in soil has been restricted to bacterial species (Dineen et al, 2010;Fang et al, 2014;Griffiths et al, 2000;Gunnigle et al, 2014;Hahn et al, 1990;Lillis et al, 2009;Mikkonen et al, 2014;Novinscak & Filion, 2011;Selenska & Klingmüller, 1992;Sessitsch et al, 2002;Sharma et al, 2004;Wang et al, 2009Wang et al, , 2012. To our knowledge, this represents the first report of the isolation of quality fungal RNA from soil.…”

Section: Stage-specific Genes Trichodermamentioning

confidence: 94%

Identification of growth stage molecular markers in Trichoderma sp. ‘atroviride type B’ and their potential application in monitoring fungal growth and development in soil

et al. 2015

View full text Add to dashboard Cite

Several members of the genus Trichoderma are biocontrol agents of soil-borne fungal plant pathogens. The effectiveness of biocontrol agents depends heavily on how they perform in the complex field environment. Therefore, the ability to monitor and track Trichoderma within the environment is essential to understanding biocontrol efficacy. The objectives of this work were to: (a) identify key genes involved in Trichoderma sp. 'atroviride type B' morphogenesis; (b) develop a robust RNA isolation method from soil; and (c) develop molecular marker assays for characterizing morphogenesis whilst in the soil environment. Four cDNA libraries corresponding to conidia, germination, vegetative growth and conidiogenesis were created, and the genes identified by sequencing. Stage specificity of the different genes was confirmed by either Northern blot or quantitative reverse-transcriptase PCR (qRT-PCR) analysis using RNA from the four stages. con10, a conidial-specific gene, was observed in conidia, as well as one gene also involved in subsequent stages of germination (L-lactate/malate dehydrogenase encoding gene). The germination stage revealed high expression rates of genes involved in amino acid and protein biosynthesis, while in the vegetative-growth stage, genes involved in differentiation, including the mitogen-activated protein kinase kinase similar to Kpp7 from Ustilago maydis and the orthologue to stuA from Aspergillus nidulans, were preferentially expressed. Genes involved in cell-wall synthesis were expressed during conidiogenesis. We standardized total RNA isolation from Trichoderma sp. 'atroviride type B' growing in soil and then examined the expression profiles of selected genes using qRT-PCR. The results suggested that the relative expression patterns were cyclic and not accumulative.

show abstract

Section: Stage-specific Genes Trichodermamentioning

confidence: 94%

Identification of growth stage molecular markers in Trichoderma sp. ‘atroviride type B’ and their potential application in monitoring fungal growth and development in soil

et al. 2015

View full text Add to dashboard Cite

show abstract

“…mRNA is not often used for the assessment of microbial community function of environmental samples due to its general instability, and difficulty in its proper extraction [42]. The study of Lünsmann et al [32] was completed in lab-scale, well controlled TWs.…”

Section: Functional Assessmentmentioning

confidence: 99%

“…This is quite a step. mRNA can be exceptionally difficult to extract from environmental samples [42]. If mRNA extraction for TWs is possible on a wider scale, micro-array and qPCR analysis could begin to return active function information.…”

Section: Conclusion and Future Horizonsmentioning

confidence: 99%

Microbial Community Assessment in Wetlands for Water Pollution Control: Past, Present, and Future Outlook

Weber

2016

Water

View full text Add to dashboard Cite

Abstract:The field of treatment wetlands (TWs) is rapidly expanding and, arguably, is tasked with studying and understanding one of the most complex water treatment systems available. Microbial communities are generally considered to be responsible for the majority of wastewater constituent degradation in TWs. However, they are also known to be spatially heterogeneous, temporally dynamic, as well as structurally and functionally diverse. Presented here is a meta-analysis of all peer reviewed TW journal articles which utilized a microbial community assessment methodology over the period of 1988 to July 2016. A total of 1101 papers were reviewed, 512 from 1988 to 2012, 215 of which included a microbial community assessment aspect and were subsequently classified as representing past research, and 589 from 2013 to July 2016, 196 of which were classified as representing current TW microbial community research. In general, TW microbial community research has increased over time, with a marked surge in the past four years. Microbial community structure is currently the most commonly used methodological type followed by activity, enumeration and function, respectively. Areas of research focus included nitrogen transformations (156), organic degradation (33), and emerging contaminants (32), with general characterization studies also accounting for a significant proportion (243). Microbial communities from a range of TW systems have been investigated over the last four years with meso-scale (10-1000 L) being the most commonly studied system size followed by large-scale (>100,000 L), micro-scale (<10 L), and pilot-scale (1000-100,000 L). Free water surface flow (SF), horizontal subsurface flow (HF), and vertical flow (VF) systems are being studied in approximately equal proportions with the majority of studies focused on gaining fixed media/biofilm samples for analysis (rather than from the rhizosphere or interstitial water). Looking at efforts from a regional perspective shows Asia to be publishing the majority of research with a main focus on VF systems and structural community assessment. European and North American studies are generally more evenly distributed among structure, function, activity, and enumeration with the majority of studies completed on HF systems. South America, Africa, and Oceania published fewer studies but focused on structural community assessment with a selection of HF, SF and VF investigations. Great strides are being made in the field of microbial community assessment in TWs with functional assessment methods being developed, better utilized, and being related directly to water treatment. The use of high-powered metagenomics sequencing such as Illumina HiSeq instrumentation is on the rise, as is the development and utilization of functional assays such as DNA microarrays and community level physiological profiling allowing for more complete community assessment. Used in concert with activity, enumeration and newly implemented stable isotope methodologies, the field of TWs is certainly moving a...

show abstract

“…Aneja et al [14] carried out random arbitrarily primed PCR (RAP-PCR) on the meta transcriptome of plant litter degrading microbial community and differential expression was successfully observed at various stages of degradation. Active populations of both eukaryotes and prokaryotes had been successfully identified by meta transcriptome based reverse transcription and sequencing of rRNA (RNA-Seq, [15]). Meta transcriptomics based study allows detection of sequences associated with a particular environmental condition that may not be so readily identified in metagenomic studies and increases the chance of detecting ecologically relevant active functions.…”

Section: Introductionmentioning

confidence: 99%

“…Several commercial kits have been made available for extracting RNA from diverse ecological samples as summarized by Wang et al [15]. Of these, MoBio RNA Power Soil Total RNA Isolation Kit has been widely used [23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Metatrascriptomic Study of Microbes in Environment: A Community Function Based Approach

Barua¹

2017

JBMOA

View full text Add to dashboard Cite

Research on microbial ecology in recent years had shifted methodologically from traditional culture based techniques to culture independent molecular techniques. This is due to the fact that it has already been established that less than 1% of microbes in environmental samples are cultivable. In recent years, metagenomics research has developed into analyzing partial or complete genomes as well as other types of molecules in environmental samples, including RNA (transcripts), proteins (translated transcripts), and metabolites (metabolic intermediates and final products). While the analyses of DNA sequences in environmental samples can provide evidence about the diversity and function of specific groups of organisms in the analyzed ecological niches, the relative importance of those genes in environmental functions cannot be determined from DNA sequences alone. To understand which genes are expressed under a specific ecological condition, the analysis of transcripts from the communities is often needed.

show abstract

Extraction of Bacterial RNA from Soil: Challenges and Solutions

Cited by 58 publications

References 151 publications

Identification of growth stage molecular markers in Trichoderma sp. ‘atroviride type B’ and their potential application in monitoring fungal growth and development in soil

Identification of growth stage molecular markers in Trichoderma sp. ‘atroviride type B’ and their potential application in monitoring fungal growth and development in soil

Microbial Community Assessment in Wetlands for Water Pollution Control: Past, Present, and Future Outlook

Metatrascriptomic Study of Microbes in Environment: A Community Function Based Approach

Contact Info

Product

Resources

About