A non-phenol–chloroform extraction of double-stranded RNA from plant and fungal tissues

Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol–chloroform extractions. In this study, an ext...

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Vydané v:	Journal of virological methods Ročník 152; číslo 1; s. 32 - 37
Hlavní autori:	Balijja, Alitukiriza, Kvarnheden, Anders, Turchetti, Tullio
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	London Elsevier B.V 01.09.2008 Amsterdam Elsevier New York, NY
Predmet:	Biological and medical sciences DNA, Fungal - isolation & purification DNA, Plant - isolation & purification dsRNA extraction dsRNA virus Fundamental and applied biological sciences. Psychology Fungi - virology Microbiology Nicotiana - virology RI-dsRNA RNA Viruses - genetics RNA, Double-Stranded - isolation & purification RNA, Viral - isolation & purification ssRNA virus replicative-intermediate Techniques used in virology Virology dsRNA virus RI-dsRNA dsRNA extraction ssRNA virus replicative-intermediate Double stranded RNA Chloroform Microbiology Phenols Method Virology
ISSN:	0166-0934, 1879-0984
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Abstract	Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol–chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing β-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method.
AbstractList	Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol–chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing β-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method. Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol-chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing beta-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method. Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol-chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing beta-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method.Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol-chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing beta-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method. Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with difficulty due to the complicated dsRNA extraction techniques used commonly which includes phenol-chloroform extractions. In this study, an extraction method for isolation of dsRNA is described that is free of phenol and chloroform. A lysis buffer, containing b-mercaptoethanol and polyvinylpolypyrrolidone (PVPP-40), was added to homogenised tissues and the subsequent supernatant was filtered through a cellulose CF-11 mini-column. DsRNA molecules were separated based on the differing affinity of nucleic acids for the cellulose CF-11 resin in 20% ethanol buffer. This easy, rapid and cheap technique has been successfully tested on fungi and plants containing different dsRNA virus molecules, indicating the possibility of a wide use of the method.
Author	Balijja, Alitukiriza Turchetti, Tullio Kvarnheden, Anders
Author_xml	– sequence: 1 givenname: Alitukiriza surname: Balijja fullname: Balijja, Alitukiriza email: balijja@ipp.cnr.it, alitukiriza@yahoo.it organization: CNR, Istituto per la Protezione delle Piante, 50019 Sesto Fiorentino, Via Madonna del Piano 10, Italy – sequence: 2 givenname: Anders surname: Kvarnheden fullname: Kvarnheden, Anders organization: Department of Plant Biology and Forest Genetics, Uppsala BioCenter SLU, Box 7080, SE-750 07 Uppsala, Sweden – sequence: 3 givenname: Tullio surname: Turchetti fullname: Turchetti, Tullio organization: CNR, Istituto per la Protezione delle Piante, 50019 Sesto Fiorentino, Via Madonna del Piano 10, Italy
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Cites_doi	10.1073/pnas.55.6.1504 10.1046/j.1365-294X.1999.00639.x 10.1080/00327487808068215 10.1016/j.jviromet.2008.01.014 10.1046/j.1439-0329.1999.00148.x 10.1139/m94-054 10.1128/AEM.65.1.287-290.1999 10.1016/j.biochi.2007.03.001 10.1016/S0166-0934(98)00079-2 10.1016/j.virusres.2003.12.002 10.1016/j.virusres.2004.12.005 10.1111/j.1439-0329.1996.tb00720.x 10.1111/j.1439-0329.1995.tb01003.x 10.1038/nrg1555 10.1006/viro.1998.9480 10.1038/nature02874 10.1016/j.chroma.2006.11.081 10.1016/0003-2697(87)90021-2 10.1094/Phyto-69-854 10.1016/0005-2787(66)90326-1 10.1146/annurev.py.28.090190.000345
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Snippet	Double-stranded RNA (dsRNA) molecules of viruses are found in nature at a very high frequency. Their detection in plants and fungi has been carried out with...
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SubjectTerms	Biological and medical sciences DNA, Fungal - isolation & purification DNA, Plant - isolation & purification dsRNA extraction dsRNA virus Fundamental and applied biological sciences. Psychology Fungi - virology Microbiology Nicotiana - virology RI-dsRNA RNA Viruses - genetics RNA, Double-Stranded - isolation & purification RNA, Viral - isolation & purification ssRNA virus replicative-intermediate Techniques used in virology Virology
Title	A non-phenol–chloroform extraction of double-stranded RNA from plant and fungal tissues
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