High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA

Abstract

Abstract. 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 (RTqPCR) 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.

A highly sensitive stem-loop RT-qPCR method to study siRNA intracellular pharmacokinetics and pharmacodynamics

“…One commonly adopted approach for siRNA extraction involves the acid-phenol: chloroform extraction followed by immobilization of RNA on glass-fiber filter. This column extraction method has been widely used for siRNA extraction [ 17 ]. Furthermore, another siRNA reverse transcription (RT) method involves the utilization of poly(A) polymerase to append a polyadenylate tail to the siRNA antisense strand, and the utilization of a poly(T) adapter as a reverse transcription primer.…”

“…As previously detailed [ 17 ], mirVana ™ PARIS ™ RNA and Native Protein Purification Kit (Thermo Fisher) were employed for siRNA extraction. The extracted siRNA was then utilized as a template for cDNA synthesis, performed using the qScript ™ microRNA cDNA Synthesis Kit (Quanta Biosciences).…”

“…While techniques such as mass spectrometry, hybridization-ELISA, and photodiode array have been proven useful for siRNA quantification, their sensitivity at the nanogram per milliliter range cannot meet the requirements for the trace amount quantification of intracellular siRNA [ 15 ]. In contrast, the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has been reported to show extremely high sensitivity for siRNA quantification, providing a more cost-effective and higher throughput way [ 16 , 17 ].…”

Quantitative contributions of processes by which polyanion drugs reduce intracellular bioavailability and transfection efficiency of cationic siRNA lipoplex

“…The extracted siRNA was quantified using our previously published RT-qPCR method [41]. Briefly, siRNA was extended using poly-A polymerase to add a poly-A tail onto the 3′-end of either strand of siRNA (qScript™ microRNA cDNA Synthesis Kit, Quanta Biosciences).…”

Plasmid DNA Isolation and Characterization Studies

“…In this study, different concentrations of plasmid DNA dissolved in (Trypsin ethylene diamine tetra acetic acid) TE were used. The absorbance values of each of the plasmid DNA samples at different concentrations were measured at 260 nm and 280 nm wavelengths in a UV visible spectrophotometer and the spectral curve equation was calculated according to the results obtained 6,14 .…”