产毒条件下层出镰刀菌总RNA提取方法的比较研究

Comparative study on the extraction methods of total RNA from Fusarium proliferatum under toxicity conditions

目的通过比较研究不同总RNA提取方法,建立一种产毒条件下层出镰刀菌总RNA提取的方法。方法样品在低温液氮高通量组织研磨仪中快速冷冻研磨,分别采用RNA提取试剂盒、Trizol法、CTAB法和改良的Trizol和试剂盒合并法提取其总RNA,并通过微量核酸检测仪、琼脂糖凝胶电泳和逆转录定量PCR(quantitative reverse transcription PCR,qRT-PCR)对提取的总RNA样品进行品质检测。结果试剂盒法不能提取到总RNA;Trizol法和CTAB法均可提取到总RNA,但质量较低,有明显降解现象;而改良的Trizol法更适合产毒条件下层出镰刀菌总RNA的提取,此方法可在30min内完成操作,获得的总RNA质量高,完整性好,条带清晰均无降解现象。结论该方法快速、方便、高效、通用,提取到的总RNA纯度高,浓度和完整性完全满足后续层出镰刀菌全转录组测序文库构建的要求。

Objective To establish a method for the extraction of total RNA from Fusarium proliferatum by comparing different total RNA extraction methods.Methods The samples were quickly frozen and ground in a high-throughput tissue grinding equipment containing low-temperature liquid nitrogen,and then the total RNA was extracted using the RNA extraction kit,Trizol method,CTAB method and modified Trizol and kit combined method.The quality of the extracted total RNA samples was tested by micro nucleic acid detector,agarose gel electrophoresis and quantitative reverse transcription PCR(qRT-PCR).Results The total RNA could not be extracted by the kit method.Both the Trizol method and the CTAB method could extract total RNA,but the quality was low,and there was obvious degradation.The modified Trizol and kit combined method was more suitable for the extraction of total RNA of F.proliferatum under toxicity conditions.This method could complete the operation within 30 min,and the total RNA obtained had high quality,good integrity and the clear bands without degradation.Conclusion This method is fast,convenient,efficient and universal.The total purity of the extracted total RNA is high,and its concentration and integrity can completely meet the requirements of the subsequent construction of the full RNA-seq library of F.proliferatum.