Figure 4

Quantification of siRNA molecules expressed from H1-shRNA, CMV-shRNA and CMV-miRNA targeting luciferase and Apolipoprotein B100. (a) Synthetic siRNA standard lines. siRNA- specific small RNA TaqMan was performed with dilution series of synthetic siLuc, siApoB1 or siApoB2 molecules. Based on molecular weight of the synthetic siLuc, siApoB1 and siApoB2, the amount of molecules for each point of standard line was calculated and plotted against CT value. (b) siLuc amplification plot (left panel) and expression in Hek293T cells (right panel). RNA was isolated two days post-transfection with 1 μg H1-shLuc, CMV-shLuc or CMV-miLuc expressing constructs and siLuc-specific small RNA TaqMan was performed. siRNA copy number was calculated using the synthetic RNA oligo standard line as described in (a) (c) siApoB1 amplification plot (left panel) and expression in Hek293T cells (right panel) after transfection with 1 μg H1-shApoB1, CMV-shApoB1 or CMV-miApoB1 expressing constructs. Experimental set up as described in (b) (d) siApoB2 amplification plot (left panel) and expression in Hek293T cells (right panel) after transfection with 1 μg H1-shApoB2, CMV-shApoB2 or CMV-miApoB2 expressing constructs. Experimental set up as described in (b). Amplification data are presented from representative experiment from two independent experiments conducted with two technical replicates. siRNA expression data are represented as mean values ± SD from 2 independent experiments conducted with two technical replicates.