3种方法提取菠萝蜜叶片和种子总RNA的比较

为了筛选最适的菠萝蜜总RNA提取方法,采用Trizol提取法、Tris-硼酸提取法和试剂盒提取法提取菠萝蜜叶片和种子总RNA,通过紫外分光光度计检测、琼脂糖凝胶电泳、RT-PCR检测以及Agilent2100检测,对提取结果进行比较分析。结果表明:采用这3种方法均能从菠萝蜜叶片和种子中提取到总RNA,Tris-硼酸提取法较其他2种方法所提取的总RNA产率高,但杂质较多;采用Tris-硼酸提取法能提取完整性较好的总RNA,28S、18S和5S清晰可见,但提取质量较试剂盒提取法差;试剂盒提取法省时省力,有明显的28S和18S条带,A260/A280分布在1.92~1.98,A260/A230分布在2.04~2.09,叶片总RNA产率42.3ng/μL,种子总RNA产率15.6ng/μL。RT-PCR检测结果表明在约200bp处存在清晰整洁的电泳条带,Agilent2100检测结果显示杂峰极少,28S峰积分面积约是18S峰积分面积的2倍。经综合评定认为,试剂盒提取法是适合菠萝蜜叶片和种子总RNA提取的方法。

Comparison of three total RNA extraction methods from leaves and seeds in Artocarpus heterophyllus

In order to screen out the optimal total RNA extracion method from Artocarpus heterophyllus,total RNA was extracted from leaves and seeds in A.heterophyllus by three methods of Trizol extraction,Tris-boric acid extraction and kit extraction.The extraction results were analyzed by UV spectrophotometer,agarose gel electrophoresis,RTPCR and Agilent 2100.The results showed that all of the three methods could extract total RNA from leaves and seeds in A.heterophyllus.Tris-boric acid extraction method had higher total RNA yield than the other two methods,but more impurities.Tris-boric acid extraction method could extract complete total RNA,28S,18S and 5S were visible and clear,but quality of extraction was inferior to that of kit extraction method.The kit extraction method was time-saving and labor-saving,extraction had distinct 28S and 18S bands,A260/A280 was from 1.92 to 1.98,A260/A230 was from 2.04 to 2.09,total RNA yield in leaves was 42.3 ng/μL,total RNA yield in seeds was 15.6 ng/μL,and there was a clear and tidy electrophoretic band at about 200 bp.The results of Agilent 2100 detection showed very few miscellaneous peak.Integral area of 28S peak was about 2 times that of 18S peak.The results of comprehensive evaluation showed that kit extraction method was suitable for total RNA extraction from leaves and seeds in A.heterophyllus.