Evaluation of commercial RNA extraction kits for the isolation of viral MS2 RNA from soil

Dineen, Shauna M.; Aranda, Roman; Dietz, Marianne; Anders, Douglas L.; Robertson, James

doi:10.1016/j.jviromet.2010.04.014

Cited by 13 publications

(14 citation statements)

References 34 publications

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“…The literature describes only one other investigation where RNA extraction was evaluated with respect to soil samples (14); however, in that study RNA extraction kits were compared utilizing soil spiked with high concentrations of virus MS2 RNA (more than 10 7 PFU g Ϫ1 soil). It is not typical for human pathogens, such as C. parvum oocysts, to be present in soil in such high concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…Kits are time efficient and have the potential to expedite and standardize sample processing and thus increase the throughput and reliability of extraction procedures. As could be expected, the selection of appropriate methods can influence the outcome of the C. parvum oocyst viability determination in terms of quality and quantity of RNA recovered (14,52,59) and the sensitivity and specificity of RT-PCR (22). The significant differences in the physical-chemical properties of various soils (i.e., organic matter, clay content, and pH) may lead to variable efficiencies of nucleic acid extraction methods (9,58).…”

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confidence: 99%

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Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA

Liang

Keeley

2011

Appl Environ Microbiol

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Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure for Cryptosporidium detection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection of Cryptosporidium with oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and Salmonella enterica serovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (␤-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed that Salmonella cells most efficiently relieved binding of RNA. With the inclusion of Salmonella during extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 ؋ 10 2 oocysts g ؊1 of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 ؋ 10 2 , 1.5 ؋ 10 3 , and 1.5 ؋ 10 4 C. parvum oocysts g ؊1 soil for sandy, loamy, and clay samples, respectively.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA

Liang

Keeley

2011

Appl Environ Microbiol

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“…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: 93%

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

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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.

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“…Commercial kits for RNA extraction from soil are also available, and are summarized in Table 1. A recent report evaluated a selection of these commercial kits (25). …”

Section: Overview Of Rna Extraction From Soilmentioning

confidence: 99%

“…For this reason, we attempted RNA extraction from Andosols with a popular commercial kit, RNA PowerSoil Total RNA Isolation Kit (MO BIO, Carlsbad, CA, USA). Unfortunately, RNA extraction failed in all Andosol soil samples tested (Wang et al , unpublished data), although this commercial kit has been proven to extract RNA from diverse soils successfully (1, 23, 25, 101, 142, 145). It is true that soil possesses detectable extracellular RNase activities (42); however, recent reports suggest that RNA could survive in the presence of extracellular RNase in soil (7, 30).…”

Section: Difficulties In Recovering Bacterial Rna From Soilmentioning

confidence: 99%

Extraction of Bacterial RNA from Soil: Challenges and Solutions

2012

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Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them.

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Evaluation of commercial RNA extraction kits for the isolation of viral MS2 RNA from soil

Cited by 13 publications

References 34 publications

Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA

Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA

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

Extraction of Bacterial RNA from Soil: Challenges and Solutions

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