A rapid and sensitive method for the estimation of individual tRNA pools |
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| Authors: | C V Patel K P Gopinathan |
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| Affiliation: | 1. State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;2. Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China;3. Key Laboratory of RNA Science and Engineering, State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China;4. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;5. Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China;6. Chongqing Innovation Institute of China Pharmaceutical University, Chongqing 401135, China;7. Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China |
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| Abstract: | ![]()
A rapid and sensitive method is described to quantitatively compare tRNA pools for individual aminoacids in a single experiment. The procedure comprises of: charging of total tRNA with a mixture of radiolabeled aminoacids, deacylation of the esterified tRNA with a volatile base and the recovery of the labeled aminoacid, derivatisation of the aminoacid with phenylisothiocyanate after mixing with excess of nonradioactive aminoacids, baseline separation of the phenylthiocarbamyl aminoacids by reverse phase high performance liquid chromatography monitored by A254nm and quantitation of the radioactivity in individual aminoacid peaks. The radioactivity in the aminoacid peak corresponds to the quantity of the aminoacylated tRNA. The method has been successfully applied to quantitate the individual tRNA pools in the developing silk glands of Bombyx mori, a functionally adapted tissue which undergoes considerable variations in tRNA content. |
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