文章基本信息

标题：Preparation of genetically engineered murine SINE RNA without endotoxin contamination
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作者：Xin Liu ; Baixue Lv ; Lifang Yan 等
期刊名称：MethodsX
印刷版ISSN：2215-0161
电子版ISSN：2215-0161
出版年度：2020
卷号：7
页码：1-7
DOI：10.1016/j.mex.2020.101102
语种：English
出版社：Elsevier
摘要：AbstractRNAs have been elucidated to play the critical role in regulating gene expression and to be expected as effective drugs in the treatment of cancer and age-related diseases. RNAs are extracted by SDS-NaCl centrifugation after transformation ofE.coliby expression vectors, which is a method to obtain genetically engineered RNAs. But the prepared RNAs by this method contain endotoxin, which limits their applicationin vivoand in cell experments. Here we improved SDS-NaCl filtration method based on SDS-NaCl centrifugation method. Endotoxin removal efficiency of SDS-NaCl filtration was nearly 4.2 times more than did SDS-NaCl centrifugation. Triton X-114 phase separation was used to reduce futher the endotoxin content of SDS-NaCI filtration-extracted RNA (from 11.25 EU/µg RNA/ml to 0.08 EU/µg RNA/ml). RNA prepared using the methods established in this paper meets the requirements forin vivoand cell culture experiments. Here we describe the process of preparing endotoxin-free B1as RNA from pET-B1as-DE3E. coli(DE3 transformed by pET-B1as expression vector which containing a tandem SINE B1 elements) using SDS-NaCl filtration incorporating Triton X-114 phase separation.•The endotoxin removal efficiency of SDS-NaCl filtration is higher than that of SDS-NaCl centrifugation.•RNA prepared by SDS-NaCl filtration incorporating Triton X-114 meets the requirements forin vivoexperiments on animals.Graphical abstractDisplay Omitted
关键词：Genetically engineered RNA;Murine SINE RNA;Endotoxin;SDS-NaCl filtration method;Triton X-114 phase separation