Preparation of genetically engineered murine SINE RNA without endotoxin contamination

MethodsX

Lifang Yan¹  Zhanjun Lv²  Murad Khan²  Suleman Shah²  Libo Su²  Ning Ji²  Xin Liu²  Xiufang Wang²  Yufang Zhao²  Zhixue Song²  Baixue Lv³ 

[1] Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022, Hubei Province, China;Department of Genetics, Hebei Key Lab of Laboratory Animal, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang 050017, Hebei Province, China;Department of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China;

关键词: Genetically engineered RNA;    Murine SINE RNA;    Endotoxin;    SDS-NaCl filtration method;    Triton X-114 phase separation;   

DOI  :  

来源: DOAJ

【 摘 要 】

RNAs 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 of E.coli by expression vectors, which is a method to obtain genetically engineered RNAs. But the prepared RNAs by this method contain endotoxin, which limits their application in vivo and 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 for in vivo and cell culture experiments. Here we describe the process of preparing endotoxin-free B1as RNA from pET-B1as-DE3 E. 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 for in vivo experiments on animals.

【 授权许可】

Unknown