Comparison and Improvement of Different Methods of RNA Isolation from Strawberry (Fragria * ananassa)

Yu, Dingqun; Tang, Haoru; Zhang, Yong; Du, Zhongjie; Yu, Haowei; Chen, Qing

doi:10.5539/jas.v4n7p51

Cited by 37 publications

(33 citation statements)

References 13 publications

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“…Extraction of total RNA from inoculated root tissue was performed similarly to the previously described 3% CTAB 3 method (Yu et al, 2012). Briefly, plant material was disrupted under liquid nitrogen in a mortar and pestle, previously decontaminated through cleaning with RNaseZap TM solution and baking for 2 h at 230 • C to deactivate RNAse enzymes.…”

Section: Inoculation Time Course Experimentsmentioning

confidence: 99%

“…Briefly, plant material was disrupted under liquid nitrogen in a mortar and pestle, previously decontaminated through cleaning with RNaseZap TM solution and baking for 2 h at 230 • C to deactivate RNAse enzymes. This material was transferred to a warmed extraction buffer (Yu et al, 2012) containing β-mercaptoethanol with 0.01 g of PVPP added per 0.1 g of frozen tissue. The remaining steps were performed as previously described (Yu et al, 2012), except that 60 µL DEPC-treated H 2 O was used to elute RNA.…”

Section: Inoculation Time Course Experimentsmentioning

confidence: 99%

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Genomic Investigation of the Strawberry Pathogen Phytophthora fragariae Indicates Pathogenicity Is Associated With Transcriptional Variation in Three Key Races

et al. 2020

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The oomycete Phytophthora fragariae is a highly destructive pathogen of cultivated strawberry (Fragaria × ananassa), causing the root rotting disease, "red core". The host-pathogen interaction has a well described gene-for-gene resistance relationship, but to date neither candidate avirulence nor resistance genes have been identified. We sequenced a set of American, Canadian, and United Kingdom isolates of known race type, along with three representatives of the closely related pathogen of the raspberry (Rubus idaeus), P. rubi, and found a clear population structure, with a high degree of nucleotide divergence seen between some race types and abundant private variation associated with race types 4 and 5. In contrast, between isolates defined as United Kingdom races 1, 2, and 3 (UK1-2-3) there was no evidence of gene loss or gain; or the presence of insertions/deletions (INDELs) or Single Nucleotide Polymorphisms (SNPs) within or in proximity to putative pathogenicity genes could be found associated with race variation. Transcriptomic analysis of representative UK1-2-3 isolates revealed abundant expression variation in key effector family genes associated with pathogen race; however, further long read sequencing did not reveal any long range polymorphisms to be associated with avirulence to race UK2 or UK3 resistance, suggesting either control in trans or other stable forms of epigenetic modification modulating gene expression. This work reveals the combined power of population resequencing to uncover race structure in pathosystems and in planta transcriptomic analysis to identify candidate avirulence genes. This work has implications for the identification of putative avirulence genes in the absence of associated expression data and points toward the need for detailed molecular characterisation of mechanisms of effector regulation and silencing in oomycete plant pathogens.

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Section: Inoculation Time Course Experimentsmentioning

confidence: 99%

Section: Inoculation Time Course Experimentsmentioning

confidence: 99%

Genomic Investigation of the Strawberry Pathogen Phytophthora fragariae Indicates Pathogenicity Is Associated With Transcriptional Variation in Three Key Races

et al. 2020

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“…Total RNA was extracted three times from 0.4 g of each sample with CTBA based extraction buffer, according to the protocol of Yu et al (2012). The quality and purity of the total RNA was evaluated by agarose gel electrophoresis and spectrometry (NanoDrop 2000, Thermo Scientific).…”

Section: Relative Gene Expression Assessed By Quantitative Rt-pcr (Rtmentioning

confidence: 99%

CO2-driven changes in energy and fermentative metabolism in harvested strawberries

Bałanda

Rosales

Palma

et al. 2015

Postharvest Biology and Technology

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“…(PVP)/β-mercaptoethanol: method indicated for strawberry tissue (Fragaria × ananassa), according to Yu et al (2012) (3% CTAB, 100 mM Tris-HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 5% PVP e 1% β-mercaptoetanol) were added to a sterile tube containing 700 µl of extraction buffer (PVP and 1% β-mercaptoethanol), homogenized and incubated at 60 °C for 10 min. The sample was centrifuged at 15,700 g for 5 min.…”

Section: ) Cetyltrimethylammonium Bromide (Ctab)/ Polyvinylpyrrolidonementioning

confidence: 99%

High-quality total RNA isolation from melon (Cucumis melo L.) fruits rich in polysaccharides

Campos

Ayub

Etto

et al. 2017

SCA

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Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazil's main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1) guanidine thiocyanate/phenol/chloroform; T2) sodium azide/β-mercaptoethanol; T3) phenol/guanidine thiocyanate; T4) CTAB/PVP/β-mercaptoethanol; T5) SDS/ sodium perchlorate/PVP/β-mercaptoethanol, and T6) sarkosyl/PVP/guanidine thiocyanate, using the AxyPrep TM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/β-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data. Key words: RNA extraction methods. RNA quality. Melon. ResumoO melão pertencente à família Cucurbitaceae é o quarto fruto mais importante no mercado mundial e a fruta mais exportada pelo Brasil. Muitas técnicas moleculares utilizadas para compreender a maturação destes frutos requerem o uso de RNA altamente purificado e em alta concentração. Entretanto, o elevado nível de compostos polifenólicos e polissacarídeos nos frutos tornam a extração de RNA um desafio. Este trabalho teve por objetivo determinar o método mais adequado para extração de RNA total em frutos de melão. Seis diferentes tampões de extração foram testados: T1) tiocianato de guanidina/ fenol/clorofórmio; T2) azida de sódio/β-mercaptoetanol; T3) fenol/tiocianato de guanidina, T4) CTAB/ PVP/β-mercaptoetanol, T5) SDS/perclorato de sódio/PVP/β-mercaptoetanol e T6) sarcosil/PVP/ tiocianato de guanidina associado com AxyPrep TM Multisource Total RNA Miniprep Kit. O melhor método para extração do RNA tanto de fruto verde quanto maduro foi o baseado no tampão SDS/

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Comparison and Improvement of Different Methods of RNA Isolation from Strawberry (Fragria * ananassa)

Cited by 37 publications

References 13 publications

Genomic Investigation of the Strawberry Pathogen Phytophthora fragariae Indicates Pathogenicity Is Associated With Transcriptional Variation in Three Key Races

Genomic Investigation of the Strawberry Pathogen Phytophthora fragariae Indicates Pathogenicity Is Associated With Transcriptional Variation in Three Key Races

CO2-driven changes in energy and fermentative metabolism in harvested strawberries

High-quality total RNA isolation from melon (Cucumis melo L.) fruits rich in polysaccharides

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