Application of a simple and affordable protocol for isolating plant total nucleic acids for RNA and DNA virus detection

Arruabarrena, Ana; Benítez-Galeano, María José; Giambiasi, Mario; Bertalmío, Ana; Colina, Rodney; Hernández-Rodríguez, Lester

doi:10.1016/j.jviromet.2016.08.011

Cited by 12 publications

(11 citation statements)

References 12 publications

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“…Most of the popular RNA isolation protocols are based on guanidine salts, such as the reagent TRIzol (Invitrogen, Carlsbad-CA-USA) and commercial kits like RNeasy (QIAGEN, Hilden-Germany) and PureLink RNA (Invitrogen, Carlsbad-CA-USA). Although these methods have been used successfully for the isolation of RNA from tissues for a variety of plants [30][31][32][33][34], for certain species, protocols based on the guanidine method have proven to be unable to isolate high quality RNA with satisfactory yields; moreover, they increase the chances of co-purifying contaminants, which interfere with downstream applications [7,35,36].…”

Section: Introductionmentioning

confidence: 99%

Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues

et al. 2021

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Acquiring high-quality RNA in sufficient amounts is crucial in plant molecular biology and genetic studies. Several methods for RNA extraction from plants are available in the literature, mainly due to the great biochemical diversity present in each species and tissue, which can complicate or prevent the extraction. Psidium guajava (Myrtaceae family) is a perennial fruit tree of medicinal and economic value; nevertheless, only a few molecular studies are available for the species. One reason is the difficulty in obtaining RNA due to the content of the samples, which are rich in polyphenols, polysaccharides, and secondary metabolites. Furthermore, there are few studies available for the isolation of RNA from guava or Psidium samples, which hampers advances in the study of the genus. Here, quality and yields of RNA isolates were compared using six extraction protocols: two protocols based on the application of cetyltrimethylammonium bromide (CTAB) lysis buffer, one protocol which uses the TRIzol reagent, one which applies guanidine thiocyanate lysis buffer followed by organic phase extraction, and two commercial kits (PureLink RNA Mini Kit and RNeasy Plant Mini Kit). The CTAB-based method provided the highest RNA yields and quality for five different tissues (flower bud, immature leaf, young leaf, mature leaf, and root), genotypes, and stress conditions. For the most efficient protocol, the average yield of RNA from guava leaves was 203.06 μg/g of tissue, and the A260/A280 and A260/A230 ratios were 2.1 and 2.2, respectively. RT-qPCR analysis demonstrated that the purity of the samples was sufficient for molecular biology experiments. CTAB-based methods for RNA isolation were found to be the most efficient, providing the highest RNA yields and quality for tissues from P. guajava. Additionally, they were compatible for downstream RNA-based applications, besides being simple and cost-effective.

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Section: Introductionmentioning

confidence: 99%

Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues

et al. 2021

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“…Pre-analysis activities are critical and reproducible, therefore, the efficient protocol is a necessity (Martinelli et al 2015). Reports have been published showing the protocols of genetic materials extraction and primers designing (Nie and Singh 2015;Zhang et al 2015;Arruabarrena et al 2016). These reports have shown the use of differential centrifugation followed by RNeasy mini kit (DCR method) to increase the efficiency of RT-PCR technique.…”

Section: Primers Design For Genetic Materials Of Potato Plants Virusesmentioning

confidence: 99%

Major In Vitro Techniques for Potato Virus Elimination and Post Eradication Detection Methods. A Review

2019

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Potato is an economically important agro-industrial crop that is conventionally propagated, however; its potential transmission of viruses through seed tubers from generation to generation is a major limitation of potato yield production. In order to produce potato virus-free and sufficient amount of potato seed tubers, several approaches of in vitro methods for virus elimination have been developed. Meristem culture has been used alone or combined with techniques such as thermotherapy, electrotherapy, cryotherapy and chemotherapy as the best alternative method for treating potato infected by viruses. Recent literature has shown that to eliminate potato virus significantly depends upon the potato cultivar, antiviral agents, type of virus, the duration of heat treatment. Appropriate duration for efficiency elimination is still under investigation. Viral elimination rate can be detected through serological methods such as enzyme-linked immunosorbent assay (ELISA) and molecular biology technique such as real time reverse transcriptase polymerase chain reaction (real time RT-PCR) that are used for pre and post elimination virus detection to evaluate the efficiency and the accuracy of virus elimination method. The purpose of this review is to highlight virus elimination methods in potato and recommending the most effective tool for virus detection in order to ensure the production of potato plantlet free of viruses. Resumen La papa es un cultivo de importancia económica agro-industrial que se propaga de manera convencional, no obstante, la potencial transmisión de virus a través del tubérculo-semilla de generación en generación es una limitación mayor en la producción del rendimiento. Con el fin de producir papa libre de virus y suficiente cantidad de tubérculo-semilla de papa, se han desarrollado varias estrategias de métodos in vitro para la eliminación de los virus. El cultivo de meristemos se ha usado solo o combinado con técnicas tales como termoterapia, electroterapia, crioterapia y quimioterapia, como el mejor método alternativo para tratar papa infectada por virus. La literatura reciente ha demostrado que para eliminar virus significativamente se depende de la variedad de papa, agentes antivirales, tipo de virus, la duración del tratamiento térmico. Aun esta bajo investigación la duración apropiada para la eliminación eficiente. El nivel de la eliminación del virus puede detectarse por métodos serológicos, tales como el inmunoensayo con enzimas conjugadas (ELISA) y técnica de biología molecular, como la reverso-transcripción de reacción en cadena de la polimerasa en tiempo real (RT-PCR) que se usan para la detección del virus pre y post eliminación para evaluar la eficiencia y la precisión del método de eliminación del virus. El propósito de esta revisión es resaltar los métodos de eliminación de virus en papa y en la recomendación de la herramienta más efectiva para la detección de virus para asegurar la producción de plántulas de papa libres de virus.

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“…The ratio of absorbance at 260 and 280 nm (A260/ A280) was at 1.82, suggesting low or no protein contamination in the nucleic acid extracts [18]. Generally, the A260/ A280 ratio at 1.8 is considered as a pure DNA [19]. Figure 1 shows a single band of extracted genomic DNA of larger than 10 kb on a 0.8% agarose gel.…”

Section: Resultsmentioning

confidence: 99%

Simple green production of silver nanoparticles facilitated by bacterial genomic DNA and their antibacterial activity

Chumpol

Siri

2017

Artificial Cells, Nanomedicine, and Biotechnology

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Green synthesis of AgNPs has gained many research interests as a low cost and eco-friendly approach. This work reported on the use of bacterial genomic DNA as the alternative biopolymer for a green production of AgNPs under a neutral pH. Although both ssDNA and dsDNA could function as stabilizing agents during the synthesis process, the ssDNA, generated by pre-heating the dsDNA at 100 °C, was more efficient to produce AgNPs with higher yield as determined by the intensity of the surface plasmon resonance (SPR) peak of silver at 475 nm. The obtained AgNPs were spherical with the average diameter of 15.0 ± 7.6 nm, which their identity was confirmed by X-ray diffraction, high-resolution transmission electron microscopy, and selected area electron diffraction analyses. The synthesized AgNPs exhibited the potent antibacterial activity against both Escherichia coli and Staphylococcus aureus with the minimum bactericidal concentrations at 250 and 500 μg/ml, respectively, suggesting their potential antibacterial activity against both Gram-negative and Gram-positive bacteria.

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Application of a simple and affordable protocol for isolating plant total nucleic acids for RNA and DNA virus detection

Cited by 12 publications

References 12 publications

Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues

Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues

Major In Vitro Techniques for Potato Virus Elimination and Post Eradication Detection Methods. A Review

Simple green production of silver nanoparticles facilitated by bacterial genomic DNA and their antibacterial activity

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