Examination of real-time polymerase chain reaction methods for the detection and quantification of modified siRNA

With the ongoing efforts to develop siRNA-based therapeutics, there is a need for high-throughput detection and quantification of siRNA. Here we report the application of four reverse-transcriptase RT-PCR-based assays for the detection of 2'-deoxy-2'-fluoro and 2'-O-methyl-modified therapeutic siRNA in mouse plasma and tissue. These assays take advantage of the dynamic range, sensitivity, specificity, and high-throughput potential found in PCR assays. Three of these assays require design and optimization of primers and/or probes specific to the siRNA while the fourth utilizes a "universal" TaqMan probe that is independent of the siRNA sequence, thereby reducing method development time and cost. For the universal assay the range of detection in mouse plasma was 500 to 5e(-5) pg/microl for four of five model Luciferase sequences tested. We found that the universal RT-PCR assay had comparable or better sensitivity and specificity than the other three assays. The universal design provides a rapid, sensitive, and specific assay with minimal method development time that will be well suited for high-throughput analysis of various siRNA sequences.