Sirna oligo duplexes

Small interfering RNA (siRNA) oligo duplexes of 27 bases in length for human PNPLA2 were purchased from OriGene Technologies, Inc. (Rockville, MD, USA). Their sequences and that of a scramble siRNA (Scr) (SR324651 and SR311349) are given in Table 3. From the six duplexes, siRNAs C, D, and E consistently provided the highest silencing efficiency; therefore, these three duplexes were used individually for silencing experiments and are referred to as siPNPLA2. ARPE-19 cells were transfected by reverse transfection in 24-well tissue culture plates as follows: A total of 6 pmol of siRNA was diluted in 100 µL of Gibco OptiMem (Thermo Fisher Scientific) per well and mixed with 1 µL of Invitrogen Lipofectamine RNAiMAX; mock transfected cells received only 1 µl of Lipofectamine. The mixture was then added to each well. After incubation at room temperature for 10 minutes, a total of 1 × 10⁵ cells in 500-µL antibiotic-free DMEM/F-12 containing 10% FBS was added to each well, and the plate was swirled gently to mix. Assays were performed 72 hours after transfection.