一种适用于RT-PCR的拮抗酵母菌总RNA提取方法

An Improved Method for Extracting Total RNA Used to RT-PCR from Antagonistic Yeast Pichia membranefaciens

为了探索提取拮抗膜醭毕赤酵母(Pichia membrane faciens)总RNA的理想方法,以拮抗膜醭毕赤酵母(Pichia membrane faciens)菌株为实验材料,用改良的SDS法、Trizol试剂法和十六烷基三乙基溴化铵(CTAB)法分别对其进行了总RNA的提取,并利用紫外分光光度计法、凝胶电泳法和反转录聚合酶链式反应(reverse transcription polymerase chainreaction,RT-PCR)比较3种不同提取方法获得的总RNA质量和产率。结果表明:改良SDS法提取的总RNA具有较高的质量,OD260/OD280比值为1.980,并且OD260/OD230为2.054。凝胶电泳结果表明,改良的SDS法有28SrRNA、18SrRNA和5SrRNA3条清晰的条带,且很少降解,28SrRNA与18SrRNA亮度比约为2:1;RT-PCR能清楚扩增到特异条带,进一步证实了改良的SDS法提取拮抗酵母膜醭毕赤菌株的总RNA质量最好;CTAB法获得的总RNA质量也较好,只是28SrRNA与18SrRNA亮度比差异不明显;Trizol试剂法提取的RNA质量最差,RNA降解多,总RNA不完整,并有DNA污染。

In order to obtain an ideal total RNA extraction method of antagonistic yeast Pichia membranefaciens, three methods, including CTAB method, Trizol method and a modified SDS method were used to extract total RNA from yeast cell and the quality and quantity were compared with ultraviolet spectrophotometer, gel electrophoresis and RT-PCR reaction. The results indicated that, the modified SDS method was better than the other methods since the value of OD260/OD280 and OD260/OD230 were 1.980 and 2.054, the purities were higher, the bands of 28S rRNA, 18SrRNA and 5S rRNA on gel electrophoresis were very clear and did not degraded, the brightness of 28S rRNA bands was twice of the bands of 18S rRNA,and the special band was amplified by RT-PCR. Comparably, CTAB method was not very good as the modified SDS method because the brightness of 28S rRNA bands was similarly as the bands of 18S rRNA. Trizol method was not fit the requirement of RNA extraction, the total RNA degraded and dispersed, DNA pollution was also found on gel electrophoresis. Therefore, the modified SDS method was appropriate for RNA extraction of antagonistic yeast and the quantity was highest.