Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio The preparation of endotoxin-free genetically engineered murine B1 antisense RNA Murad Khan a , Lifang Yan a , Baixue Lv b,c , Ning Ji a , Suleman Shah a , Xin Liu a , Zhixue Song a , Yufang Zhao a , Xiufang Wang a,* , Zhanjun Lv a,* a Department of Genetics, Hebei Medical University, Hebei Key Lab of Laboratory Animal, Shijiazhuang, 050017, Hebei Province, China b Department of Ultrasound, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, Hubei Province, China c Hubei Province Key Laboratory of Molecular Imaging, Wuhan, 430022, Hubei Province, China ARTICLE INFO Keywords: Genetically engineered RNA Murine SINE RNA Endotoxin SDS-NaCl filtration method Triton X-114 phase separation ABSTRACT 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 ge- netically 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. 1. Introduction Works in our lab and by others have shown that short interspersed nuclear elements (SINEs), which are most abundant repetitive se- quences in mammalian genomes, are involved in a multitude of cellular processes [1–5]. Alu elements are the main SINEs in human genome and B1 elements are the main SINE in the mouse genome. The head and tail tandem Alu sequences are known to suppress EGFP expression in a length-dependent manner by triggering chromatin packing via a plasmid system [6,7]. However, the transcription product of Alu (i.e., Alu RNA) plays the role in activating gene expression [8]. The B1 SINE retrotransposons regulate gene expression and have potent intrinsic insulator activity in cultured cells and live animals [9]. The small RNAs from repetitive sequences also play a fundamental role in gamete de- velopment and early individual development in mammals. Ohnishi et al. [10] reported that SINE/B1 RNAs expressed in mouse embryos and fertilized eggs before implantation were involved in the regulation of gene expression. We have previously established methods to produce genetically engineered humanized AluY RNA using BL-21 DE3 bacteria [11]. However, one of the major drawbacks to the production of genetically engineered RNA from bacteria is the contamination by endotoxin (bacterial lipopolysaccharide), which can cause fever, inflammation, cell and tissue damage, irreversible septic shock and even death in mammals [12]. Therefore, in order to study the biological functions of SINE RNAs of mouse in vivo and in vitro, one must prepare endotoxin- free SINE RNAs. There are several methods for removing endotoxin from bioengineered proteins and plasmids, including activated carbon adsorption, immobilized histidine or histamine [13], Triton X-114 phase separation [14,15], ultrafiltration [16], and more. Liu et al. [17] compared three different methods and found that Triton X-114 phase separation was the most effective way of removing endotoxin from recombinant CK-BB, CK-MB, CK-MM, myoglobin, and cardiac troponin I. Several studies have described endotoxin removal from plasmids using Triton X-114 phase separation [18,19]. RNA can be prepared using one of several methods that provide sufficient integrity, purity, and yield, including SDS-NaCl, TRIzol and SDS hot-phenol extraction. However, no study to date has reported endotoxin removal from genetically engineered RNA. In this paper, we developed a new method to prepare endotoxin-free https://doi.org/10.1016/j.ab.2020.113737 Received 11 January 2020; Received in revised form 8 April 2020; Accepted 10 April 2020 * Corresponding author. Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang, 050017, Hebei Province, China. ** Corresponding author. Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang, 050017, Hebei Province, China. E-mail addresses: wangxf1966@hebmu.edu.cn (X. Wang), lslab@hebmu.edu.cn (Z. Lv). Analytical Biochemistry 599 (2020) 113737 Available online 17 April 2020 0003-2697/ © 2020 Elsevier Inc. All rights reserved. T