Staub U, Polivka H, Gross H J
Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Würzburg, Germany.
J Virol Methods. 1995 Mar;52(1-2):209-18. doi: 10.1016/0166-0934(94)00165-d.
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.
本文描述了两种从葡萄叶片中快速微量提取总RNA的方法。将叶片组织匀浆后用苯酚提取，然后通过双水相系统结合DEAE-纤维素色谱法或用2-丁氧基乙醇溶剂进行差异沉淀来去除多酚和多糖。从0.25克葡萄叶片中得到的约5-15微克RNA质量很高，可用于逆转录、聚合酶链反应（PCR）扩增或Northern印迹分析，以检测葡萄类病毒或其他RNA。这两种方法针对在短时间内对多个样品进行小规模检测进行了优化。因此，每天可以分析12-24个叶片样品的RNA，包括逆转录和PCR扩增。这些方法的应用首次在德国葡萄中检测到了葡萄黄斑类病毒1。
