Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)

Xi Wang; Ling Zhao; Xiaoxing Wu; Huaxiu Luo; Di Wu; Meng Zhang; Jing Zhang; Mikhail Pakvasa; William Wagstaff; Fang He; Yukun Mao; Yongtao Zhang; Changchun Niu; Meng Wu; Xia Zhao; Hao Wang; Linjuan Huang; Deyao Shi; Zongyue Zeng

doi:10.1016/j.gendis.2020.04.013

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Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)

Full-length article | 更新时间：2022-06-24

- Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
- Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)
- Genes & Diseases 2021年8卷第3期
- 作者机构：
  
  1. Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory and Diagnostic Medicine, Chongqing Medical University,Chongqing,PR China,400016
  2. Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center,IL,Chicago,USA,60637
  3. Departments of Gastrointestinal Surgery, Obstetrics and Gynecology, and Nephrology, The First Affiliated Hospital of Chongqing Medical University,Chongqing,PR China,400016
  4. Department of Burn and Plastic Surgery, West China Hospital of Sichuan University,Sichuan Province,Chengdu,PR China,610041
  5. Department of Orthopaedic Surgery, The First Affiliated Hospital, Guangzhou University of Chinese Medicine,Guangdong Province,Guangzhou,PR China,510405
  6. Departments of Orthopaedic Surgery and Neurosurgery, The Affiliated Zhongnan Hospital of Wuhan University,Hubei Province,Wuhan,PR China,430072
  7. Department of Orthopaedic Surgery, The Affiliated Hospital of Qingdao University,Shandong Province,Qingdao,PR China,266061
  8. Department of Laboratory Diagnostic Medicine, Chongqing General Hospital,Chongqing,PR China,400021
  9. Institute of Bone and Joint Research, The departments of Orthopaedic Surgery and Obstetrics and Gynecology, The First and Second Hospitals of Lanzhou University,Gansu Province,Lanzhou,PR China,730030
  10. Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology,Hubei Province,Wuhan,PR China,430022
- 作者简介：
- 基金信息：
- DOI：10.1016/j.gendis.2020.04.013
  中图分类号：
- 纸质出版日期：2021，
  
  网络出版日期：2021-5-1，
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Xi Wang, Ling Zhao, Xiaoxing Wu, 等. Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)[J]. Genes & Diseases, 2021,8(3).

Xi Wang, Ling Zhao, Xiaoxing Wu, et al. Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)[J]. Genes & Diseases, 2021,8(3).
Xi Wang, Ling Zhao, Xiaoxing Wu, 等. Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)[J]. Genes & Diseases, 2021,8(3). DOI： 10.1016/j.gendis.2020.04.013.

Xi Wang, Ling Zhao, Xiaoxing Wu, et al. Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)[J]. Genes & Diseases, 2021,8(3). DOI： 10.1016/j.gendis.2020.04.013.

摘要

Abstract

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.

关键词

Keywords

DNA transfectionDNA vaccinationGene deliveryPlasmid DNA purificationRNA depletionSize selection magnetic beads

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