仙人掌总RNA提取方法的改进与分析

Improvement and analysis of cactus total RNA extraction method

利用亚硫酸钠分别与TRIzol试剂、RNA plant plus Reagent试剂、BioTeke多糖多酚植物总RNA提取试剂、艾德莱EASY spin Plus植物RNA快速提取试剂和OMEGA Plant RNA试剂结合的方法,均可获得高质量的仙人掌茎总RNA。反转录成cDNA,RT-PCR结果显示,5种方法提取的总RNA反转录后均能特异性扩增到matK基因。使用TRIzol试剂盒、RNA plant plus Reagent试剂盒和艾德莱EASY spin Plus植物RNA快速提取试剂盒提取RNA质量相对较高,在cDNA以1∶100稀释时能检测到matK基因;而使用BioTeke多糖多酚植物总RNA提取试剂盒或OMEGA Plant RNA试剂盒时,灵敏度稍低,在cDNA以1∶10稀释时,能检测到目的基因。利用亚硫酸钠结合不同植物总RNA提取试剂盒进行比较研究,为后续制备仙人掌茎高质量cDNA文库及其分子生物学研究奠定基础。

High-quality total RNAs of cactus stem were obtained by using Na：SO3 with each of TRIzol reagent, RNA plant plus reagent, BioTeke total RNA extraction reagent, EASY spin Plus plant RNA extraction reagent （ Aidlab ） or OMEGA plant RNA reagent. The eDNA RT-PCR and specific detection resuhs showed that marK gene fragments were amplified by five total RNA extraction methods. The cDNAs that reverse transcribed from the TRIzol reagent, RNA Plant Plus Reagent, and EASY spin Plus Plant RNA Extraction reagent （ Aidlab ） were relatively high, as the sensitivity PCR of eDNA highest dilution to 1 ： 100. While the cDNAs that reverse transcribed from BioTeke total RNA extraction reagent and OMEGA plant RNA reagent were relatively low, as the sensitivity RT- PCR indicated the eDNA highest dilution to 1 ： 10. In this study, Na2SO3 combined with different plant total RNA extraction kits to prepare high quality of RNA is the key of cactus stem eDNA library preparation and molecular biology.