摘要
李果实富含酚类和多糖,成熟李果实的总酚和总糖含量分别可达140.28μg/g和80.75mg/g,因此常规方法不能有效提取其RNA以进行分子生物学研究。比较了改良的异硫氰酸胍法、改良的Trizol法、改良的CTAB法和高氯酸盐法提取李果实RNA的效果,结果表明高氯酸盐法效果较好。根据李果实材料的特点,进一步优化了高氯酸盐法,包括用0.1mol/LTris-硼酸缓冲液(pH7.4)取代0.3mol/LTris-HCl缓冲液(pH8.3);提取缓冲液中β-巯基乙醇的浓度由1%提高到2%,可溶性PVP的浓度由8.5%降低至1%;联合使用10%乙醇和醋酸钾以除去多糖;用12000×g离心替代使用填充有玻璃丝的Centriflo柱实现分离提取缓冲液中杂质的目的等。在230、260和280nm下测定所提取RNA的吸光值,并用提取的RNA反转录后扩增β-actin基因片段验证RNA的质量,结果表明:用优化了的高氯酸盐法提取李果实的RNA完整性好,杂质少,得率较高,适用于RT-PCR等后续实验,并且也适合提取其他富含酚类物质或多糖果实的RNA。
Plum fruits are rich in polyphenols and polysaccharides,which may reach up to 140.28 μg/g and 80.75 mg/g,respectively,in mature fruits. As a result,routine RNA extraction methods are not suitable for producing high-grade RNA from plum fruits. In this study,several modified RNA extraction protocols were compared,including protocols utilizing guanidinium isothiocyanate,Trizol reagent,CTAB and sodium perchlorate. The RNA quality from the perchlorate method was found to be the best. The perchlorate method was further optimized,taking into account the characteristics of the plum fruit. The extraction buffer was modified by substituting 0.1 mol/L Tris-boric acid(pH 7.4) for 0.3 mol/L Tris-HCl(pH 8.3),increasing the concentration of β-mercaptoethanol from 1% to 2% and decreasing the concentration of PVP from 8.5% to 1%. In addition,ethanol and sal diureticum were added to the extraction buffer in low concentrations in order to remove polysaccharides. Finally,impurities were removed from the extraction buffer with a centrifugation step at 12 000 ×g instead of a Centriflo column filled with glass wool. The quality of the RNA extracted by this method was determined by ultraviolet spectrophotometry(at OD 230 ,OD260 and OD 280 ) and by amplifying a β-actin gene fragment with reverse transcription PCR(RT-PCR). The results indicate that plum fruit RNA extracted with the optimized perchlorate method was intact,with high yield and low concentration of impurities. It is of sufficient quality to be used in follow-up molecular biology experiments,such as RT-PCR. The improved method should be suitable for isolating RNA from other fruits with a high polyphenol or polysaccharide content.
出处
《北京林业大学学报》
CAS
CSCD
北大核心
2010年第1期57-62,共6页
Journal of Beijing Forestry University
基金
"十一五"国家科技支撑计划重点项目(2006BAD22B01)
国家自然科学基金项目(30600421)
关键词
RNA提取
高氯酸盐
李果实
多酚
多糖
RNA extraction
perchlorate
plum fruit
polyphenol
polysaccharides