Scientific Journal of KubSAU

Polythematic online scientific journal
of Kuban State Agrarian University
ISSN 1990-4665
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Name

Ushakova Yana Vladimirovna

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Academic rank

Honorary rank

Organization, job position

North Caucasian federal scientific center of horticulture, viticulture, winemaking
   

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Email

u_yana@mail.ru


Articles count: 1

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A MODIFIED PROTOCOL OF RNA ISOLATION FROM MATURE LEAVES OF GRAPES FOR RT-PCR

abstract 1431809012 issue 143 pp. 16 – 30 30.11.2018 ru 68
Isolation of high-quality RNA from the tissues of perennial woody plants, including woody grape vines, is very difficult due to the high content of phenolic compounds, secondary metabolites and polysaccharides and the ribonuclease activity of destroyed tissues. Most of the existing methods require either large time or financial costs, or do not give reproducible results in the case of RNA extraction from mature grape tissues. The modified isolation protocol is based on a combination and modification of the known RNA extraction methods, taking into account the characteristics of mature grape tissues. Existing commercial kits for the isolation of RNA from plant tissues showed a low efficiency of RNA extraction from mature grape tissues, primarily associated with "varietal specificity". Reproducible results in the extraction of RNA showed CTAB-method, however, it has several significant drawbacks associated with the duration of the extraction and the complexity of the processing of an RNA preparation with a DNAase. The developed method is based on increasing the concentration of mercaptoethanol and polyvinylpyrrolidone in the extraction buffer, eliminating the stage of RNA selective precipitation via LiCl, and replacing it with deposition on a silica-based membrane (SiO2) followed by processing with DNA-ase. and increase the purity of the preparation of RNA from genomic DNA in comparison with the original method. A modified isolation protocol was developed based on a combination and modification of known RNA extraction methods, taking into account the characteristics of mature grape tissues. This solution allows to obtain reproducible quantity and quality of RNA for the subsequent synthesis of cDNA and RT-PCR
.