An improved method for RNA extraction from common bean seeds and validation of reference genes for qPCR

Pereira, Wendell Jacinto; Bassinello, P. Z.; Brondani, C.; Vianello, R. P.

doi:10.1590/1984-70332017v17n2a22

Cited by 21 publications

(32 citation statements)

References 33 publications

(28 reference statements)

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“…Mendapatkan RNA yang berkualitas tinggi merupakan langkah awal yang sangat penting dan kritis dalam penelitian biologi molekuler. RNA dengan kualitas dan konsentrasi yang tinggi adalah tuntutan utama pada penelitian dengan teknik quantitative real-time RT PCR, konstruksi cDNA library, mikro array, analisis Northern blot, analisis transkriptomika menggunakan NGS (Next Generation Sequencing) dan lainnya (Fleige et al 2006, Schroeder et al 2006, Kiewi et al 2009, Gayral et al 2011, Pereira et al 2017.…”

Section: Pendahuluanunclassified

PERBANDINGAN TIGA KIT EKSTRAKSI RNA UNTUK ANALISIS TRANSKRIPTOMIKA PADA KELAPA SAWIT (<em>Elaeis guineensis</em> Jacq.)

Zulaeha

Purwoko

Cartealy

et al. 2019

J Bioteknol Biosains Indones

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Obtaining high-quality RNA is very important at an early stage of molecular biology research. To isolate RNA, high skill and caution are required in following laboratory procedures because RNA is easily degraded, especially samples from plant tissue culture. One of the parameters used to check the total RNA quality is RIN (RNA Integrity Number). The aim of this study was to obtain RNA extraction methods on oil palm leaves, callus and somatic embryos that were of good quality and high concentrations for transcriptomic analysis. RNA extraction was carried out using Plant RNA PureLink (Ambion), Genezol RNA Extraction (Geneaid) and RibospinTM Plant (Geneall) kit methods. The results showed that oil palm leaf, callus and somatic embryo RNA were successfully extracted using the RibospinTM (Geneall) kit. Based on the total RNA number of more than 4 μg and the RIN value of more than 7, the extracted RNA could be used in RNA sequencing for transcriptomic analysis. ABSTRAKMenghasilkan RNA berkualitas tinggi sangatlah penting pada tahap awal penelitian biologi molekuler. Untuk mengisolasi RNA diperlukan keterampilan dan kehati-hatian tinggi dalam mengikuti prosedur di laboratorium karena RNA lebih mudah terdegradasi, khususnya sampel hasil kultur jaringan tanaman. Salah satu parameter yang digunakan pada pengecekan kualitas RNA total adalah RIN (RNA Integrity Number). Penelitian bertujuan mendapatkan metode ekstraksi RNA pada daun, kalus dan embrio somatik kelapa sawit yang berkualitas baik dan memiliki konsentrasi tinggi untuk analisa transkriptomika. Ekstraksi RNA dilakukan menggunakan metode kit Plant RNA PureLink (Ambion), Genezol RNA Extraction (Geneaid) dan Ribospin TM Plant (Geneall). Hasil menunjukkan bahwa RNA daun, kalus dan embrio somatik kelapa sawit telah berhasil diekstraksi dengan menggunakan kit Ribospin TM (Geneall). RNA hasil ekstraksi tersebut dapat digunakan untuk sekuensing RNA dengan tujuan analisis transkriptomika, dilihat dari jumlah total RNA yang lebih dari 4 μg dan nilai RIN lebih dari 7.Kata Kunci: analisis RNA, embrio somatic, kalus, kelapa sawit, kualitas RNA a

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Section: Pendahuluanunclassified

PERBANDINGAN TIGA KIT EKSTRAKSI RNA UNTUK ANALISIS TRANSKRIPTOMIKA PADA KELAPA SAWIT (<em>Elaeis guineensis</em> Jacq.)

Zulaeha

Purwoko

Cartealy

et al. 2019

J Bioteknol Biosains Indones

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“…Although the use of the chaotropic agent guanidium isothiocyanate for cell lysis and denaturation of ribonucleases, chloroform to separate RNA from DNA and proteins and isopropanol for RNA precipitation was attempted, the RNA obtained from GSSC using this methodology presented high content of contaminants, as shown by the low A260/280 and A260/230 ratios obtained. 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.…”

Section: Discussionmentioning

confidence: 99%

“…Removing these contaminants is a critical step, as RNA with high purity and integrity is critical for gene expression analyses. Although there are several methods available for seed RNA extraction (Kanai et al, 2017;Pereira et al, 2017;Mornkham et al, 2013;Suzuki et al, 2004); fewer protocols are available for the extraction of good quality RNA from specific seed tissue types, especially for the seed coat (Jiang et al, 2019;Liao et al, 2019;Hong et al, 2017;Kour et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Big data from small tissues: extraction of high-quality RNA for different plant tissue types during oilseed Brassica spp. 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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“…According to Pereira et al (2017), it was used descriptive statistics to analyze these data, with emphasis on mean and standard deviation.…”

Section: Rna Quality and Cdna Librariesmentioning

confidence: 99%

“…Given the importance of RNA quality for research on gene expression, several studies are conducted to adjust RNA extraction methods (Ma et al, 2015;Pereira et al, 2017). In the case of beans, there are investigated protocols for the extraction of RNA from leaves, roots and seeds (Borges et al, 2012;Pereira et al, 2017), but few reports have been found for the stem, organ in which the symptoms of the white mold are commonly evaluated. It is believed that plant genes induced by S. sclerotiorum can be found in the transcriptome of these tissues (Oliveira et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum

et al. 2019

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The Straw Test is an assay developed to evaluate the resistance of common bean to white mold, in which the plant stems are inoculated and the symptoms of the disease are monitored. It is plausible to admit that investigating gene expression in pathogen-infected tissues may be strategically interesting. However, obtaining a quality RNA is a basic requirement for this purpose. Therefore, the objective of this study was to evaluate adjustments in protocols of commercial kits in the expectation of improving the quality of RNA obtained from bean stems. For this, plants of two lines were inoculated and the stems pathogen-infected were collected 72 hours after. For RNA extraction, two commercial reagents were used following the manufacturer’s recommendations and then following adaptations in these protocols. In particular, the proposed modifications relate to volumes of supernatant recovered in purification steps, additional step of chloroform purification and extended time for nucleic acids precipitation. The obtained RNA was analyzed by spectrophotometer, electrophoresis and bioanalyzer, then converted into cDNA and subsequently submitted to PCR. From the obtained data, it was observed that the adaptations made in the protocols contributed to better results and that, when the indicative values of RNA quality are guaranteed, the subsequent reactions are more pure, precise and representative.

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An improved method for RNA extraction from common bean seeds and validation of reference genes for qPCR

Cited by 21 publications

References 33 publications

PERBANDINGAN TIGA KIT EKSTRAKSI RNA UNTUK ANALISIS TRANSKRIPTOMIKA PADA KELAPA SAWIT (<em>Elaeis guineensis</em> Jacq.)

PERBANDINGAN TIGA KIT EKSTRAKSI RNA UNTUK ANALISIS TRANSKRIPTOMIKA PADA KELAPA SAWIT (<em>Elaeis guineensis</em> Jacq.)

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

Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum

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