Figure 6.

AGA minigene qRT-PCR splicing assay in HEK293T cells. (a) The AGA minigene construct without firefly luciferase, the qRT-PCR primers for detecting exon 2 inclusion are shown with arrows. (b,c) HEK293T cells were transfected with the minigene construct and treated with (b) 7.5 mM of the substances or (c) increasing doses of caffeine for 24 h. Total RNA was isolated, reversely transcribed and analyzed by qRT-PCR. Bars represent the mean ± SD of three independent experiments. Statistical analysis by one-way ANOVA against the untreated samples. Values of p < 0.05 were considered significant (*) while values of p < 0.001 were considered extremely significant (***). (d) The caffeine effect from luciferase assay (shown in Figure 5a) was correlated with the caffeine effect in the qRT-PCR assay shown in (c).