How Can Intracellular Full-Length siRNA Quantification Help in the Study of Carrier-Mediated Delivery In Vitro?

Colombo Scientifically Speaking continued on page 18 Figure 1. Full-length intracellular siRNA quantification protocol. (A) DSsiRNA was loaded into carriers and used for transfection of H1299-EGFP cells. (B) Total RNA was extracted and reverse transcribed (C) using stem-loop primers. Optimized RT conditions allowed for an efficient transcription of the AS. (D) The cDNA template was detected by SybrGreen qPCR, and the crossing point was calculated as the maximum of the second derivative. reproducibility by using a validated and potent siRNA suitable for in vivo research (Figure 1A); 2) H1299 cells expressing EGFP to corroborate the PCR data with flow cytometry analysis (Figure 1B); 3) an optimized reverse transcription (RT) procedure based on stem-loop primers5 (Figure 1C); and 4) SybrGreen qPCR to quantify the cDNA template (Figure 1D).