Isolation of Functional RNA From Small Amounts of Different Grape and Apple Tissues

Moser, C.; Gatto, Pamela; Moser, Mirko; Pindo, Massimo; Velasco, Riccardo

doi:10.1385/mb:26:2:95

Cited by 49 publications

(45 citation statements)

References 11 publications

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“…Unlike RNA contamination, the presence of polysaccharides and phenolic compounds interferes in the efficiency of PCR reactions and enzymatic digestion, because it inhibits the action of Taq DNA polymerase and restriction enzymes (JAPELAGHI et al, 2011;IANDOLINO et al, 2004;MOSER et al, 2004;PANDEY et al, 1996;FANG et al, 1992). Contamination by phenolic compounds is commonly observed in DNA isolation protocols (VARMA et al, 2007), since these compounds are released during cellular lysis and suffer from the action of the polyphenol oxidase enzyme, which causes oxidation of phenolic compounds when in contact with oxygen (CHOUDHARY et al, 2008;LOMMIS, 1974;MOHAMMADI et al, 2009).…”

Section: Resultsmentioning

confidence: 99%

Comparative analysis of five DNA isolation protocols and three drying methods for leaves samples of Nectandra megapotamica (Spreng.) Mez

Costa

Reiniger

Stefenon

et al. 2016

SCA

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The aim of the study was to establish a DNA isolation protocol Nectandra megapotamica (Spreng.) Mez., able to obtain samples of high yield and quality for use in genomic analysis. A commercial kit and four classical methods of DNA extraction were tested, including three cetyltrimethylammonium bromide (CTAB)-based and one sodium dodecyl sulfate (SDS)-based methods. Three drying methods for leaves samples were also evaluated including drying at room temperature (RT), in an oven at 40ºC (S40), and in a microwave oven (FMO). The DNA solutions obtained from different types of leaves samples using the five protocols were assessed in terms of cost, execution time, and quality and yield of extracted DNA. The commercial kit did not extract DNA with sufficient quantity or quality for successful PCR reactions. Among the classic methods, only the protocols of Dellaporta and of Khanuja yielded DNA extractions for all three types of foliar samples that resulted in successful PCR reactions and subsequent enzyme restriction assays. Based on the evaluated variables, the most appropriate DNA extraction method for Nectandra megapotamica (Spreng.) Mez. was that of Dellaporta, regardless of the method used to dry the samples. The selected method has a relatively low cost and total execution time. Moreover, the quality and quantity of DNA extracted using this method was sufficient for DNA sequence amplification using PCR reactions and to get restriction fragments. Key words: CTAB. SDS. Cost. Quality. Yield. ResumoO objetivo do estudo foi estabelecer um protocolo de isolamento de DNA de Nectandra megapotamica (Spreng.) Mez., capaz de obter amostras de alto rendimento e qualidade para emprego em análises genômicas. Foram testados um kit comercial e quatro métodos clássicos de extração de DNA, incluindo três métodos baseados em brometo de cetiltrimetilamônio (CTAB) e um baseado em dodecil sulfato de sódio (SDS). a secagem à temperatura ambiente (RT), em estufa a 40ºC (S40), e em forno microondas (FMO). As soluções de DNA obtidas a partir de diferentes tipos de amostras foliares utilizando os cinco protocolos foram avaliadas em termos de custo, tempo de execução e qualidade e rendimento de DNA extraído. O kit comercial não extraiu DNA com quantidade ou qualidade suficiente para o sucesso das reações de PCR. Entre os métodos clássicos, apenas os protocolos Dellaporta e Khanuja proporcionaram extração de DNA para os três tipos de amostras foliares, que resultaram em reações de PCR e ensaios com enzimas de restrição bem sucedidos. Com base nas variáveis avaliadas, o método de extração de DNA mais apropriado para Nectandra megapotamica (Spreng.) Mez. foi o Dellaporta, independentemente do método utilizado para secar as amostras. O método selecionado apresenta custo e tempo de execução total relativamente baixos. Além disso, a qualidade e quantidade do DNA extraído utilizando o método foram suficientes para amplificação de fragmentos de DNA por meio de reações de PCR e para obter fragmentos de restrição. Palavras-chave: CTAB. SDS. Cus...

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Section: Resultsmentioning

confidence: 99%

Comparative analysis of five DNA isolation protocols and three drying methods for leaves samples of Nectandra megapotamica (Spreng.) Mez

Costa

Reiniger

Stefenon

et al. 2016

SCA

View full text Add to dashboard Cite

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“…Grape seeds were removed from the berries at veraison stage by gently breaking open the berries under liquid nitrogen, then pericarp and seed portions were stored separately until use. 6 , dissolve in an appropriate volume of TE buffer or deionized water 7 , and store at -20°C. To extract total RNA, dissolve the pellet in 200 ll DEPCtreated water and follow the 9th and 10th steps.…”

Section: Plant Materialsmentioning

confidence: 99%

“…Polyphenolic compounds (particularly tannins) are readily oxidized to form covalently linked quinones, and can irreversibly bind proteins and nucleic acids to form high molecular weight complexes [2,3], whereas polysaccharides tend to co-precipitate with nucleic acids in low ionic strength buffers [4,5]. In addition, these contaminating substances severely interfere with RNA-dependent reverse transcriptase, DNA polymerase, DNA restriction endonuclease activities, and absorbance-based quantification [3,6]. In contrast to other tissues, nucleic acids extraction from fruit can be further complicated by high levels of DNase and RNase activity and substances that co-precipitate or form a covalent complex with nucleic acids [3].…”

Section: Introductionmentioning

confidence: 99%

“…So far many procedures have been published for the extraction of nucleic acids from plant tissues that vast majority are not completely satisfying because they are time-consuming [1,6], have been optimized for a specific species [7][8][9][10], are technically complex [3,5], and require ultracentrifugation steps [11,12] or the use of phenol and polyvinylpolypyrrolidone (PVPP) [12,13]. Here, we present an optimized method for nucleic acids (DNA and RNA) extraction based on the cetyltrimethylammonium bromide (CTAB)-polyvinylpyrrolidone (PVP) that in contrast to the other methods, allows efficient isolation of high quality nucleic acids from different grape tissues and fruit tissue of fruit trees.…”

Section: Introductionmentioning

confidence: 99%

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Rapid and Efficient Isolation of High Quality Nucleic Acids from Plant Tissues Rich in Polyphenols and Polysaccharides

2011

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Isolation of high quality nucleic acids from plant tissues rich in polysaccharides and polyphenols is often difficult. The presence of these substances can affect the quality and/or quantity of the nucleic acids isolated. Here, we describe a rapid and efficient nucleic acids extraction protocol that in contrast to other methods tested, effectively purify high quality nucleic acids from plant tissues rich in polysaccharides and polyphenolic compounds such as different grape tissues and fruit tissue of fruit trees. The nucleic acids isolated with this protocol were successfully used for many functional genomic based experiments including polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), cloning, and semiquantitative RT-PCR.

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“…Therefore, studies that seek to optimize the extraction conditions using reduced amount of plant material are extremely important for the advancement of molecular analyses (Moser et al, 2004;Portillo et al, 2006;Christou et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Comparative evaluation of total RNA extraction methods in Theobroma cacao using shoot apical meristems

et al. 2016

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ABSTRACT. Theobroma cacao is a species of great economic importance with its beans used for chocolate production. The tree has been a target of various molecular studies. It contains many polyphenols, which complicate the extraction of nucleic acids with the extraction protocols requiring a large amount of plant material. These issues, therefore, necessitate the optimization of the protocols. The aim of the present study was to evaluate different methods for extraction of total RNA from shoot apical meristems of T. cacao 'CCN 51' and to assess the influence of storage conditions for the meristems on the extraction. The study also aimed to identify the most efficient protocol for RNA extraction using a small amount of plant material. Four different protocols were evaluated for RNA extraction using one shoot apical meristem per sample. Among these protocols, one that was more efficient was then tested to extract RNA using four different numbers of shoot apical meristems, subjected to three different storage conditions. The best protocol was tested for cDNA amplification using reverse transcription-polymerase chain reaction; the cDNA quality was determined to be satisfactory for molecular analyses. The study revealed that with the best RNA extraction protocol, one shoot apical meristem was sufficient for extraction of high-quality total RNA. The results obtained might enable advances in genetic analyses and molecular studies using reduced amount of plant material.

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Isolation of Functional RNA From Small Amounts of Different Grape and Apple Tissues

Cited by 49 publications

References 11 publications

Comparative analysis of five DNA isolation protocols and three drying methods for leaves samples of Nectandra megapotamica (Spreng.) Mez

Comparative analysis of five DNA isolation protocols and three drying methods for leaves samples of Nectandra megapotamica (Spreng.) Mez

Rapid and Efficient Isolation of High Quality Nucleic Acids from Plant Tissues Rich in Polyphenols and Polysaccharides

Comparative evaluation of total RNA extraction methods in Theobroma cacao using shoot apical meristems

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