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Large-Scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis.
Kanwal, Fariha; Chen, Ting; Zhang, Yunlong; Zhang, Yunlon; Simair, Altaf; Rujie, Cai; Sadaf Zaidi, Najam Us Sahar; Guo, Xinhang; Wei, Xiaolong; Siegel, Geoffrey; Lu, Changrui.
Afiliación
  • Kanwal F; College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, Shanghai, China.
  • Chen T; College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, Shanghai, China.
  • Zhang Y; College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, Shanghai, China.
  • Simair A; College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, Shanghai, China.
  • Rujie C; College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, Shanghai, China.
  • Sadaf Zaidi NUS; Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan.
  • Guo X; College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, Shanghai, China.
  • Wei X; College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, Shanghai, China.
  • Siegel G; Department of Orthopaedic Surgery, Musculoskeletal Oncology Division, University of Michigan Medical School, Ann Arbor, Michigan, USA.
  • Lu C; College of Chemistry, Chemical Engineering and Biotechnology, DongHua University, Shanghai, China.
Cell Physiol Biochem ; 48(5): 1915-1927, 2018.
Article en En | MEDLINE | ID: mdl-30092596
BACKGROUND/AIMS: RNA elements such as catalytic RNA, riboswitch, microRNA, and long non coding RNA (lncRNA) play central roles in many cellular processes. Studying diverse RNA functions require large quantities of RNA for precise structure analysis. Current RNA structure and function studies can benefit from improved RNA quantity and quality, simpler separation procedure and enhanced accuracy of structural analysis. METHODS: Here we present an optimized protocol for analyzing the structure of any RNA, including in vitro transcription, size-exclusion chromatography (SEC) based denaturing purification and improved secondary structure analysis by chemical probing. RESULTS: We observed that higher Mg2+, nucleoside triphosphate (NTP) concentrations and longer reaction duration can improve the RNA yield from in vitro transcription, specifically for longer and more complicated constructs. Our improved SEC-based denaturing RNA purification effectively halved the experiment duration and labor without introducing any contaminant. Finally, this study increased the accuracy and signal-to-noise ratio (SNR) of selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing for analyzing RNA structure. CONCLUSION: Part or all of our modified method can improve almost any RNA-related study from protein-RNA interaction analysis to crystallography.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN Idioma: En Revista: Cell Physiol Biochem Asunto de la revista: BIOQUIMICA / FARMACOLOGIA Año: 2018 Tipo del documento: Article País de afiliación: China Pais de publicación: Alemania
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN Idioma: En Revista: Cell Physiol Biochem Asunto de la revista: BIOQUIMICA / FARMACOLOGIA Año: 2018 Tipo del documento: Article País de afiliación: China Pais de publicación: Alemania