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

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 (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 conc

SUBMITTER: Yeung BZ 

PROVIDER: S-EPMC4627457 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature