A modified CTAB and Trizol® protocol for high-quality RNA extraction from whole wheat seedlings, including rhizosphere

Encinas, Luis Abraham Chaparro; Arellano-Wattenbarger, Guillermo Luis; Parra-Cota, Fannie Isela; Santos-Villalobos, Sergio de los

doi:10.1007/s42976-020-00046-9

Cited by 14 publications

(9 citation statements)

References 22 publications

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“…High molarity LiCl solution precipitate RNA from DNA and favours larger transcripts precipitation, however CTAB+LiCl is a time consuming method [4]. RNA purity, defined by A260/280 ratio, is a measure of proportion between RNA and protein [2] and showed that CTAB-LiCl method provided a value of around 2 for the RNA purity, which make it a suitable method for RNA extraction.…”

Section: Resultsmentioning

confidence: 99%

Sampling for vegetative propagation: A phytosanitary status survey of grapevines collection by One Step RT-PCR method

Yzeiraj¹

2021

PVSandP

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Purpose. Grapevines (Vitis spp.) are affected by many viral diseases which cause serious pathological problems. GLRaV-3 is among the most widespread leafroll viruses, while Grapevine Fanleaf Virus (GFLV) is a destructive pathogen which reduces the lifespan of grapevine. Considering the impact and the spread of these diseases, our objective was to analyse the presence of these two viruses in several grapevine varieties in grapevine collection at ATTC Vlore. Data gathered from plant pathogens serve to better understand and prevent the spread of pathogens, as a mandatory rule for the quality control of certified plant material during vegetative propagation. Method. The presence of two common viruses were tested using virus specific primers; LC1/LC2 primer pair designed from the hHSP70 gene for detecting Grapevine Leafroll-associated Virus-3 (GLRaV3) and C3390/H2999 primer pair, designed from coat protein coding regions for detecting Grapevine Fanleaf Virus (GFLV), in six varieties; ‘Merlot’, ‘Kallmet’, ‘Shesh i zi’, ‘Shesh i bardhё’, ‘Debinё’, and ‘Pulёz’, provided through a randomised sampling procedure. One Step Reverse Transcription Polymerase Chain Reaction assay was used to detect the viral presence. Results showed a high (100%) prevalence of GLRaV3 virus in all of analysed samples, as the most frequent among the two pathogens. Analysis for of GFLV virus showed low infection rate, being present in only one sample. Conclusions. We herein show an efficient, fast and reproducible method for detecting grapevine viruses through one step RT-PCR. Our results suggest that sampling of the infected plant material should be avoided due to the presence of viral infections.

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Section: Resultsmentioning

confidence: 99%

Sampling for vegetative propagation: A phytosanitary status survey of grapevines collection by One Step RT-PCR method

Yzeiraj¹

2021

PVSandP

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“…The use of CTAB has been reported to be more suitable than SDS for the RNA extraction of samples with leafy-structures including the hard tissues of pineapple (Jordon-Thaden et al, 2015;White et al, 2008;Wong et al, 2014;Yu et al, 2012). As such, CTAB helps to break the thick lignified cell walls of the tissues, separating nucleic acids from polysaccharides (Chaparro-Encinas et al, 2020;Jaakola et al 2001;Jordon-Thaden et al, 2015). The extraction of high-quality RNA using CTAB from soft pineapple tissues was also done but was found to be unsuitable in this study; hence SDS was used to obtain total RNA.…”

Section: Discussionmentioning

confidence: 97%

Protocols for the Extraction of High-quality RNA from Pineapple Tiller, Flower, Inflorescence, and Fruits

Sehat¹,

Kumar²,

Yusuf³

2021

JTAS

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High-quality RNA is an important genetic study as it has minimal contaminants that can affect gene discovery including degraded RNAs, chemical, and biological residues. Hence, it is a prerequisite for genetic analysis using Next Generation Sequencing (NGS) for accurate and reliable data mining. Despite its importance, extracting high-quality RNA from different samples is often a challenge, as every tissue has a different biochemical composition, thus requiring different protocols. This paper reports protocols for the extraction of high-quality RNA from two type of pineapple tissues, which are thickly lignified hard tissue (tillers, inflorescence, flowers) and watery soft tissue (mature fruit, ripe fruit, and overripe fruit) via modified Kim and Hamada (2005) method. Total RNA was extracted in all six tissues, which showed two distinctive 25S and 18S band on agarose gel. The total RNA in this study was considered high-quality as the minimum concentration was 50 ng/μl, the absorbance ratio (A260:A280) was more than 1.8 and RNA integrity number (RIN) was greater than 7. The obtained results showed that the modified Kim and Hamada (2005) method was effective in extracting high-quality RNA from the challenging MD2 pineapple tissue, which is suitable for subsequent molecular analysis, including the highly sensitive NGS.

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“…The samples were then ground in a DEPC-treated mortar and stored at −80 • C until processing [50]. Total RNA was isolated using a modified TRIzol ® (Life Technologies; ThermoFisher, Waltham, MA, USA) method described by Chaparro-Encinas et al 2020 [51]. The extracted total RNA was treated with DNase I (Ambion™) to remove genomic DNA according to the manufacturer's instructions, and subsequently, RNA Integrity Number (RIN) was obtained using a BioAnalyzer Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA) to evaluate their suitability for downstream applications.…”

Section: Rna Extraction and Rna-seq Analysismentioning

confidence: 99%

Transcriptional Regulation of Metabolic and Cellular Processes in Durum Wheat (Triticum turgidum subsp. durum) in the Face of Temperature Increasing

Encinas

Santoyo

Peña‐Cabriales

et al. 2021

Plants

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The Yaqui Valley, Mexico, has been historically considered as an experimental field for semiarid regions worldwide since temperature is an important constraint affecting durum wheat cultivation. Here, we studied the transcriptional and morphometrical response of durum wheat at an increased temperature (+2 °C) for deciphering molecular mechanisms involved in the thermal adaptation by this crop. The morphometrical assay showed a significant decrease in almost all the evaluated traits (shoot/root length, biovolume index, and dry/shoot weight) except in the dry root weight and the root:shoot ratio. At the transcriptional level, 283 differentially expressed genes (DEGs) were obtained (False Discovery Rate (FDR) ≤ 0.05 and |log2 fold change| ≥ 1.3). From these, functional annotation with MapMan4 and a gene ontology (GO) enrichment analysis with GOSeq were carried out to obtain 27 GO terms significantly enriched (overrepresented FDR ≤ 0.05). Overrepresented and functionally annotated genes belonged to ontologies associated with photosynthetic acclimation, respiration, changes in carbon balance, lipid biosynthesis, the regulation of reactive oxygen species, and the acceleration of physiological progression. These findings are the first insight into the regulation of the mechanism influenced by a temperature increase in durum wheat.

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A modified CTAB and Trizol® protocol for high-quality RNA extraction from whole wheat seedlings, including rhizosphere

Cited by 14 publications

References 22 publications

Sampling for vegetative propagation: A phytosanitary status survey of grapevines collection by One Step RT-PCR method

Sampling for vegetative propagation: A phytosanitary status survey of grapevines collection by One Step RT-PCR method

Protocols for the Extraction of High-quality RNA from Pineapple Tiller, Flower, Inflorescence, and Fruits

Transcriptional Regulation of Metabolic and Cellular Processes in Durum Wheat (Triticum turgidum subsp. durum) in the Face of Temperature Increasing

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