Islas-Flores Ignacio, Peraza-Echeverría Leticia, Canto-Canché Blondy, Rodríguez-García Cecilia Mónica
Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Calle 43 No. 130 Chuburná de Hidalgo, Mérida, Yucatán, 97200, México.
Mol Biotechnol. 2006 Sep;34(1):45-50. doi: 10.1385/MB:34:1:45.
High-quality RNA preparations are critical for further applications such as reverse transcriptase-polymerase chain reaction (RT-PCR) transcript amplifications, and elaboration of cDNA and expressed sequence tag libraries. Melanins are phenolic compounds present in many fungi and apparently play key roles in fungi pathogenesis and survival. However, during RNA extraction these compounds constitute a significant challenge to extraction of substantial quantities of high-quality RNA, and consequently to preparation of cDNA libraries. No method currently exists for RNA extraction from Mycosphaerella fijiensis that produces high quantities of melanin-free RNA. This fungus is the most important pathogen of cultivated Musa sp. varieties. A comparison is made between results obtained from the Trizol and RNeasy protocols for RNA extraction, two commercially available methods commonly used to obtain RNA from various sources. An improved methodology is described that allows isolation of intact RNA and elimination of melanins from M. fijiensis mycelium. RNA quality is evaluated by electrophoresis in formaldehyde-agarose gels, RT into cDNAs, and subsequent PCR amplification using primers designed against actin and beta- tubulin from fungi.
高质量的RNA制剂对于诸如逆转录聚合酶链反应（RT-PCR）转录扩增以及构建cDNA和表达序列标签文库等进一步应用至关重要。黑色素是存在于许多真菌中的酚类化合物，显然在真菌致病和生存中起关键作用。然而，在RNA提取过程中，这些化合物对提取大量高质量RNA构成了重大挑战，从而对cDNA文库的制备也造成了挑战。目前尚无从香蕉黑条叶斑病菌中提取无黑色素的高质量RNA的方法。该真菌是栽培香蕉品种最重要的病原体。对从Trizol和RNeasy RNA提取试剂盒这两种常用的从各种来源获取RNA的商业方法所获得的结果进行了比较。本文描述了一种改进的方法，该方法可以从香蕉黑条叶斑病菌菌丝体中分离出完整的RNA并去除黑色素。通过在甲醛-琼脂糖凝胶中进行电泳、逆转录成cDNA以及随后使用针对真菌肌动蛋白和β微管蛋白设计的引物进行PCR扩增来评估RNA质量。
