High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA.
RNAi therapeutics provide an opportunity to correct faulty genes, and several RNAi have entered clinical evaluation. The existing quantification methods typically use radioactivity- or fluorescence-labeled RNAi, require large blood volumes, and/or have a limited dynamic detection range. We established a quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assay to measure RNAi; the model analyte was survivin siRNA (siSurvivin). A second siRNA was used as the internal standard. The three major steps were (a) extraction of the two siRNAs from blood or water, (b) synthesis of their cDNA by poly-A extension, and (c) qPCR of cDNA. Standard curves were established. Utility of the assay was demonstrated in a pharmacokinetic study where all 12 samples for the blood concentration-time profile were obtained from a single mouse given an intravenous dose of 1 nmole siSurvivin (prepared as lipoplex with pegylated cationic liposomes). The RT-qPCR assay was sensitive (lower detection limit of 100 fM) and had a 5 × 107-fold dynamic range and low sample volume requirement (10 μL). The 16-point standard curves constructed using whole blood samples were linear (R (2) > 0.98). The intraday and interday variations for the slopes were ≤6%, although the variations for accuracy and precision at individual concentrations were substantially higher (58-145%). Standard curves prepared with water in place of blood showed similar results (<6% difference), indicating water may be used when blood is not available. The current RT-qPCR assay enabled the measurement of nonlabeled siRNA in small volume of blood samples.
Department of Pharmaceutical Sciences, University of Oklahoma, Oklahoma City, Oklahoma, 73117, USA.
Bertrand Z
BZ
Yeung
Optimum Therapeutics LLC, 1815 Aston Ave, Suite 107, Carlsbad, California, 92008, USA.
Ze
Z
Lu
Optimum Therapeutics LLC, 1815 Aston Ave, Suite 107, Carlsbad, California, 92008, USA.
Guillaume M
GM
Wientjes
Department of Pharmaceutical Sciences, University of Oklahoma, Oklahoma City, Oklahoma, 73117, USA. jau@optimumtx.com.|Optimum Therapeutics LLC, 1815 Aston Ave, Suite 107, Carlsbad, California, 92008, USA. jau@optimumtx.com.|Taipei Medical University, Taipei, Taiwan, Republic of China. jau@optimumtx.com.
Jessie L-S
JL
Au
D000818:Animals; D000925:Anticoagulants / Q000097:blood / Q000493:pharmacokinetics; D001810:Blood Volume / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D051034:Inhibitor of Apoptosis Proteins / Q000097:blood* / Q000493:pharmacokinetics*; D057230:Limit of Detection; D051379:Mice; D008819:Mice, Nude; D034741:RNA, Small Interfering / Q000097:blood* / Q000493:pharmacokinetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012097:Repressor Proteins / Q000097:blood* / Q000493:pharmacokinetics*; D000077022:Survivin
D000925:Anticoagulants; C117749:Birc5 protein, mouse; D051034:Inhibitor of Apoptosis Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D000077022:Survivin
RT-qPCR; blood; pharmacokinetics; siRNA