The preparation of endotoxin-free genetically engineered murine B1 antisense RNA
The preparation of endotoxin-free genetically engineered murine B1 antisense RNA
复制标题
无内毒素基因工程鼠B1反义RNA的制备
DOI：
10.1016/j.ab.2020.113737
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发表时间：
2020-06-15
影响因子：
2.9
通讯作者：
Lv, Zhanjun
中科院分区：
文献类型：
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作者：
Khan, Murad;Yan, Lifang;Lv, Zhanjun
从大肠杆菌（E. coli）中生产基因工程RNA的主要限制之一是内毒素污染。在这里，我们报告了第一种能够从基因工程RNA中去除内毒素的方法。为了证明这一概念，我们用含有小鼠基因组串联短穿插核元件（sinb1元件）的质粒转化了大肠杆菌。然后，我们评估了几种提取方法（SDS- nacl离心、SDS- nacl过滤、TRIzol和SDS热酚）及其改进方法，并测量了所得RNA产率、RNA纯度、RNA完整性和内毒素含量。以2 mol/L NaCl进行SDS-NaCl过滤，以DEPC作为RNA保护剂，可有效去除内毒素，获得较好的RNA产率。Triton X-114相分离进一步降低了SDS-NaCl过滤提取RNA的内毒素含量。SDS-NaCl滤过、Triton X-114相分离提取的RNA在BALB/c小鼠中未引起不良反应，家兔注射后未引起发热。RNA符合动物体内实验核酸试剂的要求。
One of the major limitations in the production of genetically engineered RNA from Escherichia coli (E. coli) is contamination by endotoxin. Here we report the first method that is capable of removing endotoxin from genetically engineered RNA. As a proof of concept, we transformed E. coli with a plasmid containing a tandem short interspersed nuclear elements from the mouse genome (SINE B1 elements). We then evaluated several extraction methods (SDS-NaCl centrifugation, SDS-NaCl filtration, TRIzol and SDS hot-phenol) and refinements thereof, and measured the resulting RNA yield, RNA purity, RNA integrity and endotoxin content. SDS-NaCl filtration with 2 mol/L NaCl, incorporating DEPC as an RNA protective agent, effectively removed endotoxin and resulted in a good RNA yield. Triton X-114 phase separation further reduced the endotoxin content of SDS-NaCl filtration-extracted RNA. RNA extracted by SDS-NaCl filtration with Triton X-114 phase separation did not cause adverse reactions in BALB/c mice and did not induce fever in rabbits when injected into these animals. The RNA met the requirements of nucleic acid reagents for in vivo experiments on animals.