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KMID : 0613820190290060653
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Journal of Life Science
2019 Volume.29 No. 6 p.653 ~ p.661
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Validation of Stem-loop RT-qPCR Method on the Pharmacokinetic Analysis of siRNA Therapeutics
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Kim Hye-Jeong
Kim Taek-Min
Kim Hong-Joong
Jung Hun-Soon
Lee Seung-Ho
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Abstract
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The first small interfering RNA (siRNA) therapeutics have recently been approved by the Food and Drug Administration in the U.S., and the demand for a new RNA therapeutics bioanalysis method?which is essential for pharmacokinetics, including the absorption, distribution, metabolism, and excretion of siRNA therapeutics?is rapidly increasing. The stem-loop real-time qPCR (RT-qPCR) assay is a useful molecular technique for the identification and quantification of small RNA (e.g., micro RNA and siRNA) and can be applied for the bioanalysis of siRNA therapeutics. When the anti-HPV E6/E7 siRNA therapeutic was used in preclinical trials, the established stem-loop RT-qPCR assay was validated. The limit of detection was sensitive up to 10 fM and the lower limit of quantification up to 100 fM. In fact, the reliability of the established test method was further validated in three intra assays. Here, the correlation coefficient of R2>0.99, the slope of -3.10~-3.40, and the recovery rate within ±20% of the siRNA standard curve confirm its excellent robustness. Finally, the circulation profiles of siRNAs were demonstrated in rat serum, and the pharmacokinetic properties of the anti-HPV E6/E7 siRNA therapeutic were characterized using a stem-loop RT-qPCR assay. Therefore, the stem- loop RT-qPCR assay enables accurate, precise, and sensitive siRNA duplex quantification and is suitable for the quantification of small RNA therapeutics using small volumes of biological samples.
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KEYWORD
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Bioanalysis, pharmacokinetics, siRNA therapeutics, stem-loop RT-qPCR, validation
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