| Autor: |
Xin Liu, Baixue Lv, Lifang Yan, Murad Khan, Ning Ji, Suleman Shah, Zhixue Song, Yufang Zhao, Libo Su, Xiufang Wang, Zhanjun Lv |
| Jazyk: |
angličtina |
| Rok vydání: |
2020 |
| Předmět: |
|
| Zdroj: |
MethodsX, Vol 7, Iss , Pp 101102- (2020) |
| Druh dokumentu: |
article |
| ISSN: |
2215-0161 |
| DOI: |
10.1016/j.mex.2020.101102 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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