Wang Xi, Zhao Ling, Wu Xiaoxing, Luo Huaxiu, Wu Di, Zhang Meng, Zhang Jing, Pakvasa Mikhail, Wagstaff William, He Fang, Mao Yukun, Zhang Yongtao, Niu Changchun, Wu Meng, Zhao Xia, Wang Hao, Huang Linjuan, Shi Deyao, Liu Qing, Ni Na, Fu Kai, Hynes Kelly, Strelzow Jason, El Dafrawy Mostafa, He Tong-Chuan, Qi Hongbo, Zeng Zongyue
Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory and Diagnostic Medicine, Chongqing Medical University, Chongqing, 400016, PR China.
Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, 60637, USA.
Genes Dis. 2020 May 20;8(3):298-306. doi: 10.1016/j.gendis.2020.04.013. eCollection 2021 May.
Plasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, purification of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A. To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps. Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Mag-bind-based SSMB method costs only 5-10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.
从细菌细胞中分离质粒DNA（pDNA）是分子克隆和生物医学研究中最常见且关键的步骤之一。几乎所有的pDNA纯化都涉及细菌的裂解、膜脂、蛋白质和基因组DNA的去除、从大量裂解物中纯化pDNA以及浓缩pDNA以供下游应用。虽然使用了许多液相和固相pDNA纯化方法，但最终的pDNA制品通常会受到不同程度的宿主RNA污染，而这些RNA无法被核糖核酸酶A完全消化。为了开发一种简单、经济高效且有效的RNA去除方法，我们研究了市售的大小选择磁珠（SSMB），如Mag-Bind® TotalPure NGS试剂盒（或Mag-Bind），是否能完全去除pDNA制品中的细菌RNA。在这项原理验证研究中，我们证明，与核糖核酸酶A消化和两种商业质粒亲和纯化试剂盒相比，SSMB方法在从pDNA小量制备物中去除污染RNA方面效率极高。基因转染和细菌集落形成试验表明，通过SSMB方法纯化的pDNA在质量和完整性方面优于通过核糖核酸酶A消化和/或商业质粒纯化试剂盒纯化的pDNA样品。我们进一步证明，SSMB方法能完全去除大规模pDNA样品中的污染RNA。此外，基于Mag-bind的SSMB方法在每个样品的成本上仅为大多数商业质粒纯化试剂盒的5-10%。因此，所报道的SSMB方法可以成为一种用于常规pDNA制备中去除细菌RNA的有价值且廉价的工具。
