Two rapid microscale procedures for isolation of total RNA from leaves rich in polyphenols and polysaccharides: application for sensitive detection of grapevine viroids.
Two rapid microscale procedures for isolation of total RNA from grapevine leaves are described. Homogenized leaf tissue is phenol extracted and polyphenols and polysaccharides are removed by either an aqueous two-phase system followed by DEAE-cellulose chromatography or by differential precipitation with the solvent 2-butoxyethanol. The resulting RNA, about 5-15 micrograms from 0.25 g grapevine leaves, is of high quality and can be employed for reverse transcription and polymerase chain reaction (PCR) amplification or Northern blot analysis to detect grapevine viroids or other RNAs. The two procedures are optimized for small-scale examination of many samples in a short time. Thus RNA from 12-24 leaf samples can be analyzed per day, including reverse transcription and PCR amplification. The application of these procedures led to the first detection of grapevine yellow speckle viroid 1 in German grapevines.
Staub
U
U
Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Würzburg, Germany.
Polivka
H
H
Gross
H J
HJ
D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D017931:DNA Primers; D005026:Ethylene Glycols; D005419:Flavonoids; D005638:Fruit / Q000821:virology*; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D010636:Phenols; D018515:Plant Leaves; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011108:Polymers; D059808:Polyphenols; D011134:Polysaccharides; D012367:RNA, Viral / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012997:Solvents; D014772:Viroids / Q000302:isolation & purification*
D017931:DNA Primers; D005026:Ethylene Glycols; D005419:Flavonoids; D010636:Phenols; D011108:Polymers; D059808:Polyphenols; D011134:Polysaccharides; D012367:RNA, Viral; D012997:Solvents; C017096:n-butoxyethanol