考察了环磷酸腺苷(cyclic adenosine monphosphate,cAMP)生产酵母菌株G2在YPD培养基中不同生长阶段的总RNA提取效果,并进行了初步改进研究。选择16 h对数生长期、36 h二次生长期和60 h稳定期细胞,分别用试剂盒提取,RNA得率依次为1 010.6、171.5和91.3 ng/A600。选择高、低丰度表达基因ACT1和CYR1进行实时荧光定量反转录聚合酶链式反应(quantitative real-time reverse transcription polymerase chain reaction,qRT-PCR),同时判断DNA污染情况;琼脂糖电泳和RNA完整值(RNA integrity number,RIN)测定判断完整性。结果显示,36 h和60 h RNA提取物都存在DNA污染,仅16 h样品能满足RNA测序要求。进一步对比了5种破壁方法在破碎细胞及相应RNA提取上的效果,添加珠打破壁步骤破碎细胞效果最好,显示对16 h细胞RNA提取效果没有明显变化,但36 h和60 h细胞的RNA得率分别提高至979.6和785.6 ng/A600,为前述值的5.71倍和8.60倍,且纯度能满足qRT-PCR要求,36 h样品能满足RNA测序要求。该研究有助于推动建立酵母生长全阶段RNA分析技术和深化cAMP相关途径调控机制的认识。
In this study, the yield and quality of total RNA extracted from whole course cultures of a 3′,5′-cyclic adenosine monophosphate (cAMP) - producing yeast strain G2 in yeast extract peptone dextrose medium was investigated and further optimization was carried out. The cells with culture time of 16, 36 and 60 h were selected, which corresponded to exponential, post-diauxic and stationary phase, respectively. Using a commercial RNAout kit, the RNA yields were obtained as 1 010.6, 171.5 and 91.3 ng/A600, respectively. The genes ACT1 and CYR1 were selected as the relatively high and low representatives, respectively, and used in quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR) and DNA pollution analysis. Agarose gel electrophoresis and RNA integrity number (RIN) test were used in RNA integrity assay. The results showed that 36 h and 60 h RNA extractions were polluted with DNA and only 16 h sample was qualified for RNA-Seq. Five cell wall disruption methods were further selected to investigate the disruption ratio and their effects on RNA extraction. The addition of bead-beating was proved to have no significant effect on the 16 h cells, while it increased the RNA yields of the 36 h and 60 h cells to 979.6 and 785.6 ng/A600, which were 5.71- and 8.60- fold of the value before modification, respectively. Further, all samples were qualified for qRT-PCR and 36 h isolations also met the requirement of RNA-Seq. This study helps to establish the RNA extraction protocol of yeast suitable for the whole course cultures and provides valuable insights into the mechanism relating to the cAMP pathway regulating.
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