Preparation of genetically engineered murine SINE RNA without endotoxin contamination

MethodsX
Xin LiuZhanjun Lv

Abstract

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 hig...Continue Reading

References

Aug 8, 2007·Biotechnology and Bioengineering·Aleksei RozkovStefan R Schmidt
Mar 1, 2012·Analytical Biochemistry·Rui MaLi-Wen Li
Feb 11, 2016·Critical Reviews in Biotechnology·Elvina Clarie Dullah, Clarence M Ongkudon
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