Development of Pre-Clinical Models for Evaluating the Therapeutic Potential of Candidate siRNA Targeting STAT6
Figure 2
Optimisation of the bio-analytical assay.
(a-d): Maintenance of bio-analytical assay sensitivity within different biological matrices. 372u or 372 m siRNA were spiked into 1 × TE buffer (•), plasma (□) or rat lung tissue extract (▵) at a range of concentrations (0.032 – 100 pg/µl) and then analysed using an RT-PCR-based bio-analytical assay. No significant difference in the threshold cycle of detection (Ct) was noted between the three matrices at any of the siRNA concentrations of tested. Shown are representative standard curves obtained for sense (a, b) and anti-sense (c, d) strand measurement. Values presented are mean (n = 3) with errors bars omitted for clarity. Percent PCR efficiency for 372u (sense) in TE buffer, plasma, or lung extract = 94.2%, 86.3% and 102.8% respectively. Percent PCR efficiency for 372 m (sense) = 124.2%, 101.5%, 114.8% respectively. (e, f): To compare the effect of RNA extraction methods on bio-analytical assay sensitivity, rat lung tissue was spiked with 372u (e) or 372m (f) (125 ng) and homogenised in 4M guanidine isothiocyanate (GITC) or Tri-reagent. Tri-reagent homogenised tissue was processed through the manufacturer’s recommended procedure for the extraction of RNA, either up to the chloroform phase separation step (TR-AQ) or through the entire process (TR-F). Recovery of siRNA from TR-AQ processed tissue was comparable to GITC, recovery from TR-F processed tissue was significantly lower. Values presented are mean ± S.E.M (n = 3), and represent % siRNA recovered in comparison to GITC.
