Fig. 1.

Gene-silencing effects of siRNA candidates targeting mTOR in HeLa cells. a mTOR mRNA and protein levels were measured by real-time RT-PCR (upper panel) and immunoblot analysis (lower panel). HeLa cells were transfected with mTOR-targeting siRNA or control siRNA at 100 nM using Oligofectamine. After 24 h, total RNA was isolated for RT, or whole-cell lysates were prepared for immunoblot analyses (n = 3). Real-time PCR was carried out using iQ SYBR Green Supermix as described in “Materials and methods.” b mTOR protein levels were monitored at 24 and 48 h. Band densities were analyzed with a densitometer (LAS-4000, n = 3). c To detect specific target mTOR mRNA cleavage, modified 5′RACE designed to amplify cleaved mTOR mRNA as final PCR product was performed (n = 2). Total RNA from cells transfected with the corresponding siRNAs was ligated to the GeneRACER RNA adaptor. Sequence-specific mRNA cleavage by mTOR siRNA was confirmed by both the presence of 210-bp PCR products as indicated by arrow and sequencing analysis for this PCR product. Each value represents the mean ± SD. *p < 0.05, **p < 0.01