Microamp fast 0.1 ml 96 well reaction plates

HeLa-d1-eGFP cells were seeded 1 × 10⁵ cells per well in a 12-well plate and incubated for 24 h prior to siRNA treatment with siGFP-1 or negative control siRNA. Serial dilution of siRNA, lipoplex formulation and treatment were carried out as described above. Negative control siRNA was prepared at the same concentration as the highest siGFP dose. After 24 h lipoplex incubation, cells were washed with PBS, and RNA was extracted using GenElute Mammalian Total RNA Miniprep Kit according to the manufacturer’s protocol. Complementary DNA was obtained using SuperScript III First-Strand Synthesis System (Sigma) with random hexamer primers running on a MasterCycler EpGradient 5341 thermal cycler (Eppendorf AG, Hamburg, Germany). Real-Time qPCR was performed on a StepOnePlus Real-Time PCR System using MicroAmp Fast 0.1 mL 96-well Reaction Plates (Applied Biosystems, Foster City, CA, USA) and SYBR Green JumpStart Taq Readymix (Sigma) for the qPCR reactions. GAPDH was used as a housekeeping gene, data were analyzed with StepOne Software v.2.3, and d1-eGFP expression fold-change relative to the control sample was calculated using the ∆∆Ct method.

HeLa-d1-eGFP or HeLa wild-type cells were incubated with 40 nM chol-siGFP (d1-eGFP), 200 nM chol-siPPIB, or chol-siGAPD (wild type), or control chol-siRNA at the same concentrations, prepared OptiMEM. After 6 h, the medium was removed and cells were washed once with PBS and incubated with complete DMEM supplemented with the indicated drugs for 18 h. At the end of the experiment, samples were washed once with PBS, and RNA extraction was performed using GenElute Mammalian Total RNA Miniprep Kit (Sigma) according to the manufacturer’s protocol. SuperScript III First-Strand Synthesis System (Sigma) was used for complementary DNA synthesis with random hexamer primers, and performed on an Mastercycler EpGradient 5341 thermal cycler (Eppendorf AG, Hamburg, Germany). SYBR Green Jumpstart Taq Readymix (Sigma) was used for qPCR reactions, and performed on a StepOnePlus Real-Time PCR System with MicroAmp Fast 0.1 mL 96-well Reaction Plates (Applied Biosystems, Foster City, CA, USA). GAPD or ACTB was used as house-keeping genes for normalization. Samples treated with control chol-siRNA with or without any additional drug treatment was used as calibrator samples. Fold differences compared to controls was calculated using the ΔΔC_t method. Data were analyzed with StepOne Software v.2.3.