A general method for high-quality RNA isolation from metabolite-rich fruits

Gudenschwager, Orianne; González-Agüero, Mauricio; Defilippi, Bruno G.

doi:10.1016/j.sajb.2012.08.004

Cited by 44 publications

(38 citation statements)

References 27 publications

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“…Total RNA from grape samples was isolated using the hot borate method (Gudenschwager et al, 2012). The first strand of cDNA was obtained by carrying out reverse transcription reactions with 2 g of total RNA as a template, using MMLV-RT reverse transcriptase (Promega, Madison, WI, USA) and oligo dT primers (Invitrogen, Breda, The Netherlands).…”

Section: Rna Isolation and Cdna Synthesismentioning

confidence: 99%

Study on differential expression of 1-aminocyclopropane-1-carboxylic acid oxidase genes in table grape cv. Thompson Seedless

Muñoz-Robredo

Gudenschwager

Chervin

et al. 2013

Postharvest Biology and Technology

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International audienceAs a consequence of the non-climacteric status of grapes (Vitis vinifera), ethylene biosynthesis and signal transduction have scarcely been studied in this fruit. In spite this drawback, the available information suggests a role for ethylene in ripening grape berries. In this work, we report the identification of three homologous genes that encode 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), a key component of ethylene biosynthesis. A comparison of protein sequences revealed that all three VvACOs harbor a 2OG-Fe(II) oxygenase domain, which is typical of the ACO gene family; however, VvACO1 showed a higher amino acid sequence homology with VvACO2 than with VvACO3. The expression pattern of VvACOs and the effect of exogenous ethylene on their transcript accumulation were evaluated during table grape berry development in the "Thompson Seedless" cultivar. A peak in VvACO1 transcript accumulation levels was registered around veraison that was 4-fold higher than at harvest, and this peak was confirmed during a second season in grapes that were harvested from three different vineyards. An enhancement in ethylene production and VvACO genes transcript levels was observed in grapes sprayed with ethephon during berry development. However, VvACO1 transcripts reached the highest accumulation earlier than VvACO2 and VvACO3. Altogether, these data confirmed that ethylene may have a role in some aspects of the grape ripening process, and they also highlighted the potential use of some VvACO genes as molecular markers for identifying grape veraison stages in grapes

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Section: Rna Isolation and Cdna Synthesismentioning

confidence: 99%

Study on differential expression of 1-aminocyclopropane-1-carboxylic acid oxidase genes in table grape cv. Thompson Seedless

Muñoz-Robredo

Gudenschwager

Chervin

et al. 2013

Postharvest Biology and Technology

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“…For other seed species with high levels of polyphenols, RNA obtained using TRIzol reagent was also degraded and presented low A260/230 ratios (Pereira et al, 2017;Mornkham et al, 2013), suggesting that TRIzol is not a good methodology to extract RNA from seeds or seed coats which contain these secondary metabolites. Although hot borate has been described as a good protocol for extracting RNA from samples rich in polysaccharides, phenolic compounds and oil (Gudenschwager et al, 2012: Wan & Wilkins., 1994, the RNA extracted from MSSC with this method was not pure. MSSC accumulate high amounts of polyphenols in the maturation stage of seed development, specially oxidised procyanidins which confer the dark brown colour of the seed coat characteristic of this stage of development.…”

Section: Discussionmentioning

confidence: 99%

Big data from small tissues: extraction of high-quality RNA for different plant tissue types during oilseedBrassicaspp. seed development for RNA-Sequencing

Siles

Eastmond

Kurup

2019

Preprint

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Obtaining high-quality RNA for gene expression analyses from different seed tissues is challenging due to the presence of various contaminants, such as polyphenols, polysaccharides and lipids which interfere with RNA extraction methods. At present, the available protocols for extracting RNA from seeds require high amounts of tissue and are mainly focused on extracting RNA from whole seeds.However, extracting RNA at the tissue level enables more detailed studies regarding tissue specific transcriptome during development. Seeds from heart stage embryo to mature developmental stages of Brassica napus and B. oleracea were sampled for isolation of the embryo, endosperm and seed coat tissues. Ovules and gynoecia wall tissue were also collected at the pre-fertilization stage. After testing several RNA extraction methods, E.Z.N.A. Plant RNA Kit and Picopure RNA Isolation kit extraction methods with some modifications, as well as the use of PVPP for seed coats and endosperms at green stages, resulted in high RNA concentrations with clear 28S and 18S bands and high RIN values. Here, we present efficient and reliable RNA extraction methods for different genotypes of Brassica spp for different tissue types during seed development. The high-quality RNA obtained by using these methodologies is suitable for RNA-Sequencing and gene expression analyses.Abbreviations: HSE: Heart stage embryo, HSEnd: heart stage endosperm, HSSC: heart stage seed coat, TSE: torpedo stage embryo, TSSC: torpedo stage seed coat, TSEnd: torpedo stage endosperm, GSE: green stage embryo, GSEnd: green stage endosperm, GSSC: green stage seed coat, MSE: mature stage embryo, MSSC: mature stage seed coat.

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“…Total RNA was isolated from 1.0 g frozen tissue using a modified hot borate method [45], following all the indications listed in the cited protocol with the only exception that sample tissue was reduced to a third of the recommended amount and polyvinylpyrrolidone was doubled to avoid phenolic compound contamination before RNA isolation. The quantity and quality of RNA were assessed with Qubit® 2.0 fluorometer (Invitrogen™ by Life Technologies, Singapore).…”

Section: Rna Isolationmentioning

confidence: 99%

Transcriptomic study of pedicels from GA3-treated table grape genotypes with different susceptibility to berry drop reveals responses elicited in cell wall yield, primary growth and phenylpropanoids synthesis

et al. 2020

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Background: Gibberellins (GA 3 ) are the most sprayed growth regulator for table grape production worldwide, increasing berry size of seedless varieties through pericarp cell expansion. However, these treatments also exacerbate berry drop, which has a detrimental effect on the postharvest quality of commercialized clusters. Several studies have suggested that pedicel stiffening caused by GA 3 would have a role in this disorder. Nevertheless, transcriptional and phenotypic information regarding pedicel responses to GA 3 is minimal.Results: Characterization of responses to GA 3 treatments using the lines L23 and Thompson Seedless showed that the former was up to six times more susceptible to berry drop than the latter. GA 3 also increased the diameter and dry matter percentage of the pedicel on both genotypes. Induction of lignin biosynthesis-related genes by GA 3 has been reported, so the quantity of this polymer was measured. The acetyl bromide method detected a decreased concentration of lignin 7 days after GA 3 treatment, due to a higher cell wall yield of the isolated fractions of GA 3treated pedicel samples which caused a dilution effect. Thus, an initial enrichment of primary cell wall components in response to GA 3 was suggested, particularly in the L23 background. A transcriptomic profiling was performed to identify which genes were associated with these phenotypic changes. This analysis identified 1281 and 1787 genes differentially upregulated by GA 3 in L23 and cv. Thompson Seedless, respectively. Concomitantly, 1202 and 1317 downregulated genes were detected in L23 and cv. Thompson Seedless (FDR < 0.05). Gene ontology analysis of upregulated genes showed enrichment in pathways including phenylpropanoids, cell wall metabolism, xylem development, photosynthesis and the cell cycle at 7 days post GA 3 application. Twelve genes were characterized by qPCR and striking differences were observed between genotypes, mainly in genes related to cell wall synthesis. Conclusions: High levels of berry drop are related to an early strong response of primary cell wall synthesis in the pedicel promoted by GA 3 treatment. Genetic backgrounds can produce similar phenotypic responses to GA 3 , although there is considerable variation in the regulation of genes in terms of which are expressed, and the extent of transcript levels achieved within the same time frame.

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A general method for high-quality RNA isolation from metabolite-rich fruits

Cited by 44 publications

References 27 publications

Study on differential expression of 1-aminocyclopropane-1-carboxylic acid oxidase genes in table grape cv. Thompson Seedless

Study on differential expression of 1-aminocyclopropane-1-carboxylic acid oxidase genes in table grape cv. Thompson Seedless

Big data from small tissues: extraction of high-quality RNA for different plant tissue types during oilseedBrassicaspp. seed development for RNA-Sequencing

Transcriptomic study of pedicels from GA3-treated table grape genotypes with different susceptibility to berry drop reveals responses elicited in cell wall yield, primary growth and phenylpropanoids synthesis

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