A RAPID AND EFFECTIVE METHOD FOR TOTAL RNA EXTRACTION FROM CONIDIA AND MYCELIUM OF SEPTORIA TRITICI

Journal of Rapid Methods and Automation in Microbiology Pub Date : 2011-01-19 DOI:10.1111/j.1745-4581.2003.tb00408.x

JIAN-RONG GUO, KAMEL A. ABD-ELSALAM, FRANK SCHNIEDER, JOSEPH-ALEXANDER VERREET

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引用次数: 2

Abstract

Abstract An effective and rapid method for RNA extraction from conidia and mycelium of S. tritici was developed, in which guanidinium isothiocyanate, CTAB and insoluble PVP were combined in one step, simplifying the extraction process greatly. The procedure can be completed in less than 6 h. Yield and purity of RNA could be estimated by electrophoresis on 1% formaldhyde-denatured agarose gel and by measuring the absorbance at 280, 260, 230 and 210 nm by UV spectroscopy. The result of electrophoresis showed two clear bands of 18S and 28S rRNA and no DNA contamination in the isolated RNA solution. The ratios A260/A280, A260/A230, A260/A210 were 1.76–1.82, 2.79–3.21 and 0.81–0.86, respectively. A high yield with 532–881 ng total RNA per gram fresh conidia and mycelium of S. tritici was obtained. The RNA was of sufficient purity for RT-PCR to detect the expression of β-tubulin gene in S. tritici. This protocol may also be suitable for RNA extraction from spores and mycelium of other fungi.

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一种快速有效地提取小麦隔孢孢子和菌丝体总RNA的方法

摘要采用异硫氰酸胍、CTAB和不溶性PVP一次性联合提取小麦黑麦草分生孢子和菌丝体中RNA的方法，大大简化了提取过程。在1%甲醛变性琼脂糖凝胶上电泳，并在280、260、230和210 nm处测定吸光度，可以估计RNA的产率和纯度。电泳结果显示18S和28S两条清晰的rRNA条带，分离的RNA溶液中没有DNA污染。A260/A280、A260/A230、A260/A210的比值分别为1.76 ~ 1.82、2.79 ~ 3.21和0.81 ~ 0.86。麦黑麦鲜分生孢子和菌丝体的总RNA为532 ~ 881 ng / g，产量较高。该RNA具有足够的纯度，可用于RT-PCR检测小麦β-微管蛋白基因的表达。该方法也适用于其他真菌孢子和菌丝体的RNA提取。

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Journal of Rapid Methods and Automation in Microbiology 生物-生物工程与应用微生物

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