Validation of Stem-loop RT-qPCR Method on the Pharmacokinetic Analysis of siRNA Therapeutics

Stem-loop RT-qPCR 분석법을 이용한 siRNA 치료제의 생체시료 분석법 검증 및 약물 동태학적 분석

  • 김혜정 (인핸스드바이오) ;
  • 김택민 (인천대학교 생명공학부 나노바이오전공) ;
  • 김홍중 (인핸스드바이오) ;
  • 정헌순 (인핸스드바이오) ;
  • 이승호 (인천대학교 생명공학부 나노바이오전공)
  • Received : 2019.05.07
  • Accepted : 2019.05.28
  • Published : 2019.06.30


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 $R^2$>0.99, the slope of -3.10 ~ -3.40, and the recovery rate within ${\pm}20%$ 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 stemloop 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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Fig. 1. Schematic description of stem-loop RT-qPCR.

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Fig. 2. Specific amplification of Anti-HPV E6/E7 siRNA using stem loop RT-qPCR assay.

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Fig. 3. Determination of low limit of quantification (LLOQ) concentration.

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Fig. 4. Reproducibility and linearity of stem-loop RT-qPCR assay for quantitative detection of siRNA (A-C).

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Fig. 5. Quantitative detection of Anti-HPV E6/E7 siRNA in rat serum.

Table 4. Precision of stem-loop RT-qPCR from independent experiments

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Table 5. Pharmacokinetic parameter analysis of Anti-HPV E6/ E7 siRNA in rat serum

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Table 1. Sequences of siRNA, stem-loop primer and RT-qPCR primers used in the study

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Table 2. Stem-loop RT-qPCR results with serial diluted standard siRNA. Comparison of mean Cp value (thrreee replicates) obtained with stem-loop RT-qPCR

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Table 3. Robustness of stem-loop RT-qPCR from different researchers

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Supported by : 중소기업청


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