Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts

Nassuth, Annette; Pollari, Elisa; Helmeczy, Katherine; Stewart, Sandra; Kofalvi, S. A.

doi:10.1016/s0166-0934(00)00211-1

Cited by 134 publications

(64 citation statements)

References 21 publications

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“…The reports view, modify buffer may improve the extraction of leaves and buds in RNA content and purity to some extent [22,23]. In this author Li Z [10] and Heath et al [11] reported improved method for space mutation sunflower flower bud RNA extraction.…”

Section: Electrophoresis Test Results and Analysismentioning

confidence: 97%

An Optimized Preparation Method to Obtain High-quality RNA from the Buds of Sunflowers

Feng¹,

Ma²,

Yang³

2016

Proceedings of the 2016 5th International Conference on Environment, Materials, Chemistry and Power Electronics

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Abstract.In an attempt to isolate high-quality, total RNA from flower buds of sunflowers (Helianthus annuus). We have already improved the SDS method. After adding liquid nitrogen, we have to grind the material down. After that we add protein -DNA precipitation, wash it with the addition of different kinds of alcohol, such as 75% alcohol and precipitation. Then we add DEPC water and guanidine hydrochloride blend, LiCl overnight precipitation,and then wash it off with 75% alcohol, finally make it dissolved in 15 µL DEPC water which collocated in minus 80 environment for storage. The total RNA is precipitated with 1/10 volume of NaAc and 2 volumes of absolute ethanol to prevent contamination by polysaccharides. These experimental results show that the total RNA extracted by this method is of high purity and integrity, and the electrophoretic bands are clear. D260/D230, D260/D280 and RNA (g/100mg) are 2.05, 2.11 and 9.52, respectively. Total RNA can amplify the target gene fragment by PT-PCR. This improved method can be used to extract the high-quality RNA from the sunflower buds of the space mutation, in order to study the molecular mechanism of the next step.

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Section: Electrophoresis Test Results and Analysismentioning

confidence: 97%

An Optimized Preparation Method to Obtain High-quality RNA from the Buds of Sunflowers

Feng¹,

Ma²,

Yang³

2016

Proceedings of the 2016 5th International Conference on Environment, Materials, Chemistry and Power Electronics

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“…Pemilihan metode ekstraksi RNA terutama yang mampu meminimalkan sejumlah inhibitor reverse transcriptase dan/atau polymerase dalam ekstrak tanaman sehingga akan menghalangi deteksi virus meskipun virus ada dalam ekstrak (Azizi et al, 2017). Polivynil pirolidone dengan polifenol komplek (John, 1992) dan/atau merkaptoetanol dapat berperan sebagai sebuah antioksidan, digunakan secara bersama dalam sebuah prosedur ekstraksi oleh beberapa peneliti dalam usaha untuk mengurangi level inhibitor (Nassuth et al, 2000) seperti halnya polisakarida atau polifenol dalam jaringan tanaman (Newbury & Possingham, 1977;Sahu et al, 2012;John, 1992). Menurut Li et al (2008) RNeasy Plant Mini Kits (Qiagen) tidak sesuai untuk tanaman yang kandungan polifenol dan polisakarida tinggi serta menghasilkan konsentrasi RNA Turnip mosaic virus rendah dengan kemurnian tinggi untuk mengekstraksi benih Brassica rappa (Adiputra et al, 2012).…”

Section: Pengantarunclassified

Perbaikan Metode Ekstraksi dsRNA Virus secara Sederhana untuk RT-PCR Tiga Virus Tumbuhan

Endarsih

Hartono

Sulandari

2018

JPTI

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Replicative form (RF) INTISARIReplicative form (RF) virus RNA merupakan asam nukleat berstruktur dsRNA, selalu ditemukan pada tumbuhan terinfeksi oleh virus RNA. Prinsip kerja ekstraksi dsRNA berdasarkan afinitas serbuk selulosa terhadap asam nukleat dan adsorbsi spesifik dsRNA pada konsentrasi etanol 16,6 %. Penelitian ini bertujuan untuk mengembangkan metode ekstraksi dsRNA secara sederhana untuk preparasi deteksi RT-PCR terhadap Rehmannia mosaic virus (ReMV), Cucumber mosaic virus (CMV), Tomato chlorosis virus (ToCV) dan dibandingkan dengan kit komersil. Data yang dibandingkan adalah kuantitas asam nukleat, analisa efisiensi metode (tingkat kerumitan, waktu, biaya per reaksi) serta sekuensing. Konsentrasi RNA hasil ekstraksi metode dsRNA secara sederhana lebih rendah dibanding dengan metode kit, namun kedua metode menghasilkan RNA yang murni. Berdasarkan hasil PCR dan sekuensing disimpulkan bahwa virus penyebab mosaik daun lada dan tembakau serta klorosis daun tomat berturut-turut adalah CMV, ReMV, dan ToCV dengan ukuran amplikon berturut turut 500, 568 dan 360 pb. Metode ini cukup murah dan kuantitas RNA yang dihasilkan sebanding dengan kit komersil. Ekstraksi RNA menggunakan metode dsRNA secara sederhana dapat dikembangkan untuk preparasi deteksi RT-PCR terhadap CMV, ReMV, ToCV.Kata kunci: ekstraksi dsRNA, kuantitas RNA, Rehmannia mosaic virus, RT-PCR, serbuk selulosa

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“…Enzyme Linked Immunosorbant Assay (ELISA) is convenient and easy method but it has a limitation of being less sensitive when the viral load is low (Landgraf et al, 1991). In this context, Nucleic acid based polymerase chain reaction (PCR) (Edwards and Gibbs, 1994;Grieco and Gallitelli, 1999;Tettelin et al, 1999;Olmos et al, 2002;Stellrecht et al, 2004) and hybridization methods have the advantage of amplifying the target nucleic acid present even at very low level and it has become an attractive technique for the diagnosis of plant viral diseases (Mullis et al, 1986;Henson and French, 1993;Xie and Hu, 1994;McManus and Jones, 1995;Hafner et al, 1997a and b;Elnifro et al, 2000;Nassuth et al, 2000).…”

Section: Issn: 2319-7706 Volume 6 Number 6 (2017) Pp 398-411mentioning

confidence: 99%

Banana Bunchy Top Viral Coat Protein (CP) Gene Expression Studies at Molecular Level in Hill Banana cv. Sirumalai (AAB)

Arumugam¹,

Kalaimughilan²,

Kathithachalam³

2017

Int.J.Curr.Microbiol.App.Sci

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Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts

Cited by 134 publications

References 21 publications

An Optimized Preparation Method to Obtain High-quality RNA from the Buds of Sunflowers

An Optimized Preparation Method to Obtain High-quality RNA from the Buds of Sunflowers

Perbaikan Metode Ekstraksi dsRNA Virus secara Sederhana untuk RT-PCR Tiga Virus Tumbuhan

Banana Bunchy Top Viral Coat Protein (CP) Gene Expression Studies at Molecular Level in Hill Banana cv. Sirumalai (AAB)

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