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Broad application of a simple and affordable protocol for isolating plant RNA.

ABSTRACT: Standard molecular biological methods involve the analysis of gene expression in living organisms under diverse environmental and developmental conditions. One of the most direct approaches to quantify gene expression is the isolation of RNA. Most techniques used to quantify gene expression require the isolation of RNA, usually from a large number of samples. While most published protocols, including those for commercial reagents, are either labour intensive, use hazardous chemicals and/or are costly, a previously published protocol for RNA isolation in Arabidopsis thaliana yields high amounts of good quality RNA in a simple, safe and inexpensive manner.We have tested this protocol in tomato and wheat leaves, as well as in Arabidopsis leaves, and compared the resulting RNA to that obtained using a commercial phenol-based reagent. Our results demonstrate that this protocol is applicable to other plant species, including monocots, and offers yield and purity at least comparable to those provided by commercial phenol-based reagents.Here, we show that this previously published RNA isolation protocol can be easily extended to other plant species without further modification. Due to its simplicity and the use of inexpensive reagents, this protocol is accessible and affordable and can be easily implemented to work on different plant species in laboratories worldwide.

SUBMITTER: Couto D

PROVIDER: S-EPMC4404699 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Project description:Finding standard cost-effective methods for monitoring biodiversity is challenging due to trade-offs between survey costs (including expertise), specificity, and range of applicability. These trade-offs cause a lack of comparability among datasets collected by ecologists and conservationists, which is most regrettable in taxonomically demanding work on megadiverse inconspicuous taxon groups. We have developed a site-scale survey method for diverse sessile land organisms, which can be analyzed over multiple scales and linked with ecological insights and management. The core idea is that field experts can effectively allocate observation effort when the time, area, and priority sequence of tasks are fixed. We present the protocol, explain its specifications (taxon group; expert qualification; plot size; effort) and applications based on >800 original surveys of four taxon groups; and we analyze its effectiveness using data on polypores in hemiboreal and tropical forests. We demonstrate consistent effort-species richness curves and among-survey variation in contrasting ecosystems, and high effectiveness compared with casual observations both at local and regional scales. Bias related to observer experience appeared negligible compared with typical assemblage variation. Being flexible in terms of sampling design, the method has enabled us to compile data from various projects to assess conservation status and habitat requirements of most species (specifically rarities and including discovery of new species); also, when linked with site descriptions, to complete environmental assessments and select indicator species for management. We conclude that simple rules can significantly improve expert-based biodiversity surveys. Ideally, define (i) a common plot size that addresses multiple taxon groups and management goals; (ii) taxon groups based on field expertise and feasible number of species; (iii) sufficient and practical search time; (iv) a procedure for recording within-plot heterogeneity. Such a framework, combined with freedom to allocate effort on-site, helps utilizing full expertise of observers without losing technical rigor.

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Broad application of a simple and affordable protocol for isolating plant RNA.

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