Dose and time dependence of transient RNAi silencing of PCS1 in protoplasts. A, Protoplasts were transfected with 1, 5, or 10 μg of dsRNA against PCS1 (RNAi) or with water (Control) and incubated for 24 h in the dark. Silencing of PCS1 in dsRNA- and mock-transfected protoplasts was analyzed by RT-PCR. The representative RT-PCR results of three independent transfection events are shown. B, Relative abundance of PCS1 transcript on the gel in A. Relative abundance was normalized to the level of the transcript of ACT2 using GelQuant software. C, RT-PCR analysis of the time dependence of RNAi silencing of PCS1 in protoplasts. Protoplasts were transfected with 10 μg of dsRNA against PCS1 (RNAi) or with water (Control). Control and RNAi protoplast samples were collected at indicated time points and analyzed by RT-PCR for the abundance of PCS1 transcript. ACT2 was amplified as a control for amount of a template.