siRNA Serum Stability

Serum stability of both free siRNA and modified siRNA complexes before and after nebulization was investigated by incubating each formulation at 37 ºC with equal volume of human serum to give 50% serum concentration. At each predetermined time interval, (0, 5, 15, 30min, 1, 2, 3, 4, 5, 6, 7, 24 and 48 h) 50 μL of the mixture were removed and stored at -20ºC until gel electrophoresis was performed. In order to release siRNA from the complexes for gel electrophoresis, each sample was treated with 25 mM of reduced glutathione and 100 μM of PMAA. The aliquots from different incubation time periods were loaded onto 4% NuSieve 3:1 Reliant agarose gels in 1×TBE buffer (0.089 M Tris/Borate, 0.002 M EDTA, pH 8.3; Research Organic Inc., Cleveland, OH) and subjected to submarine electrophoresis. The gels were stained with EtBr, digitally photographed, and scanned using Gel Documentation System 920 (NucleoTech, San Mateo, CA).