Suitability of six extraction methods for isolating a large quantity of high-quality RNA from New Zealand free-draining stony soil

Podolyan, Anastasija; Grelet, Gwen‐Aëlle

doi:10.1080/00288233.2020.1753785

Cited by 2 publications

(1 citation statement)

References 12 publications

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“…Yet, we lack a deep understanding of how soil microbial community composition and functions support and influence soil biogeochemical processes. A variety of environmental DNA (eDNA) sequencing methods have been employed to gain unprecedented insight into soil microbial community composition and diversity (Podolyan and Grelet 2021; Wang et al 2009c). For example, amplicon-based gene sequencing (Aguiar-Pulido et al 2016; Zuñiga et al 2017) and shotgun metagenomics (Azeem et al 2021; Shakya et al 2019) are widely used for deeper investigation of the soil microbiome.…”

Section: Introductionmentioning

confidence: 99%

Soil metatranscriptomics: An improved RNA extraction method toward functional analysis using nanopore direct RNA sequencing

Poursalavati

Javaran

Laforest‐Lapointe

et al. 2022

Preprint

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Soil microbes play an undeniable role in sustainable agriculture, plant health, and soil management. A deeper understanding of soil microbial composition and function has been gained through next-generation sequencing. While soil metagenomics has provided valuable information about microbial diversity, issues stemming from RNA extraction, low RNA abundance in some microbial populations (e.g., viruses), and mRNA enrichment have slowed the progress of soil metatranscriptomics. A variety of soil RNA extraction methods have been developed so far. Yet none of the available protocols can obtain RNA with high quality, purity, and yield for third-generation sequencing. This latter requires RNA with high quality and large quantities (with no or low contamination, such as humic acids). Also, use of commercial kits for in-batch soil RNA extraction is quite expensive, and these commercial kits lack buffer composition details, which prevents the optimization of protocols for different soil types. An improved and cost-effective method for extracting RNAs from mineral and organic soils is presented in this paper. An acidic sodium acetate buffer and phosphate buffer with modifications to bead-beating and nucleic acid precipitation lead to higher RNA yields and quality. Using this method, we obtained almost DNA-free RNA. By using nanopore's direct RNA sequencing, the extracted contamination-free RNAs were successfully sequenced. Lastly, taxonomic groups such as bacteria, fungi, archaea, and viruses were classified and profiled as well as functional annotation of the datasets was carried out using an in-house customized bioinformatics workflow.

show abstract

Section: Introductionmentioning

confidence: 99%

Soil metatranscriptomics: An improved RNA extraction method toward functional analysis using nanopore direct RNA sequencing

Poursalavati

Javaran

Laforest‐Lapointe

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

Soil Metatranscriptomics: An Improved RNA Extraction Method Toward Functional Analysis Using Nanopore Direct RNA Sequencing

Poursalavati

Javaran

Laforest‐Lapointe

et al. 2023

Phytobiomes Journal

View full text Add to dashboard Cite

Soil microbes play an undeniable role in sustainable agriculture, plant health, and soil management. A deeper understanding of soil microbial composition and function has been gained through next-generation sequencing. While soil metagenomics has provided valuable information about microbial diversity, issues stemming from RNA extraction, low RNA abundance in some microbial populations (e.g., viruses), and mRNA enrichment have slowed the progress of soil metatranscriptomics. A variety of soil RNA extraction methods have been developed so far. Yet none of the available protocols can obtain RNA with high quality, purity, and yield for third-generation sequencing. The latter requires RNA with high quality and large quantities (with no or low contamination, such as humic acids). Also, use of commercial kits for in-batch soil RNA extraction is quite expensive, and these commercial kits lack buffer composition details, which prevents the optimization of protocols for different soil types. An improved and cost-effective method for extracting RNAs from mineral and organic soils is presented in this paper. An acidic sodium acetate buffer and phosphate buffer with modifications to bead-beating and nucleic acid precipitation lead to higher RNA yields and quality. Using this method, we obtained almost DNA-free RNA. By using nanopore’s direct RNA sequencing, the extracted contamination-free RNAs were successfully sequenced. Lastly, taxonomic groups such as bacteria, fungi, archaea, and viruses were classified and profiled as well as functional annotation of the datasets was carried out using an in-house customized bioinformatics workflow.

show abstract

Suitability of six extraction methods for isolating a large quantity of high-quality RNA from New Zealand free-draining stony soil

Cited by 2 publications

References 12 publications

Soil metatranscriptomics: An improved RNA extraction method toward functional analysis using nanopore direct RNA sequencing

Soil metatranscriptomics: An improved RNA extraction method toward functional analysis using nanopore direct RNA sequencing

Soil Metatranscriptomics: An Improved RNA Extraction Method Toward Functional Analysis Using Nanopore Direct RNA Sequencing

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