金柑果肉组织总RNA提取方法比较

Comparison of extraction methods of total RNA from Fortunella japonica pulp tissue

为给高通量测序等对RNA质量要求较高的后续试验提供基础,以‘桂金柑一号’等5个金柑品种的果实为材料,采用异硫酸胍提取法、CTAB提取法、SDS提取法、试剂盒提取法提取金柑果实总RNA,并对所提取的总RNA浓度、A260/A280、A260/A230、凝胶电泳结果、RT-PCR扩增产物等指标进行比较分析。结果表明:CTAB提取法、SDS提取法和试剂盒提取法能较好地去除金柑果实中的蛋白质、金柑甙、柠檬萜、DNA、多酚、多糖、单宁、色素等大分子物质以及盐类等小分子杂质。采用异硫酸胍提取法所提取的总RNA浓度最高,达到95.9μg/g,其A260/A280在1.69～1.73之间,A260/A230> 2.14;其它RNA提取方法所提取的总RNA浓度最高达到76.9μg/g,其A260/A280分布在1.88～1.97之间,A260/A230分布在2.00～2.09之间,符合高质量RNA的要求范围。除了异硫酸胍提取法所提取的RNA琼脂糖电泳结果未见清晰的5S条带,其余方法所提取的RNA经琼脂糖电泳均可以观察到清晰的28S、18S、5S条带。但采用异硫酸胍提取法、CTAB提取法、SDS提取法和试剂盒提取法所提取的金柑果实总RNA进行反转录均可以得到清晰的扩增产物。在4种提取方法中,采用CTAB提取法所提取金柑果实总RNA浓度较高,纯度也较理想,琼脂糖电泳和RT-PCR效果好,是进行后续相关研究的较佳选择。

In order to provide some basic for carrying out the follow-up experiments of high throughput sequencingrequiring high quality of RNA and so on,taking fruits from five kumquat cultivars including'Gui Kumquat No.1'asmaterials,total RNA in kumquat fruits was extracted by the methods of guanidine isosulfate extraction,CTAB extraction,SDS extraction and kit extraction,and total RNA concentration,A260/A280,A260/A230,gel electrophoresis results and RTPCRamplification products were compared and analyzed.The results showed that the methods of CTAB extraction,SDS extraction and kit extraction could better remove small molecular impurities,such as protein,fortunellin,lemonterpene,DNA,polyphenols,polysaccharides,tannins,pigments,salt,and other large and small molecular substances.The concentration of total RNA extracted by guanidine isosulfate extraction method was the highest,reaching95.9μg/g.thevalues of A260/A280were from1.69to1.73,A260/A230>2.14.The concentrations of total RNA extracted by the otherRNA extraction methods could be up to76.9μg/g,and the values of A260/A280were distributed from1.88to1.97,and thevalues of A260/A230were from2.00to2.09,which conformed to request scope of high quality of RNA.No clear5S bandwas found in RNA agarose electrophoresis extracted by the guanidine isoguanidine extraction method.Some clear28S,18S and5S bands from the RNA extracted by the other methods were observed by agarose electrophoresis.However,clear amplification products of the total RNA from kumquat fruit extracted by the methods of guanidine isosulfate extraction,CTAB extraction,SDS extraction and kits extraction could be obtained through reverse transcription.In thefour extraction methods,the total RNA concentration by the CTAB extraction method was higher,the purity was better,agarose electrophoresis and RT-PCR had better effects,and it was better choice for sub sequent related researches.