In vitro A-to-I editing-specific cleavage of ribozyme against FLNA mRNA extracted from cells. (A) Experimental procedure for analyzing editing-specific cleavage of HR-FLNA-edit against extracted FLNA mRNA by measuring the editing ratio at the Q/R site. Total RNAs containing FLNA mRNA were extracted from Tet-ADAR2 cells and then reacted with HR-FLNA-edit to measure editing-specific cleavage. The editing ratio was calculated from the sequence chromatogram generated by direct sequencing of the RT–PCR product from the ribozyme-reacted total RNA. (B) Sequence chromatograms of products with the cleavage reaction of HR-FLNA-edit and HR-FLNA-mut, or in the absence of HHR. Chromatograms, which were generated using reverse FLNA-specific primers, are shown for the sequences obtained from the reaction with HR-FLNA-edit for 3 h (top, right) and 24 h (bottom, right), and from the incubation without ribozyme for 3 h (top, left) and 24 h (bottom, left) that were used as controls. (C) Quantification of the editing ratio of FLNA mRNA after the ribozyme reaction. The bar graph shows the editing ratios at the Q/R site from the HR-FLNA-edit reaction (dark gray bars), HR-FLNA-mut (light gray bars), and without reaction (white bars). Editing ratios were quantified by measuring the peak heights of T and C and calculated on the basis of the peak height of C divided by C+T. Results are presented as an average editing ratio and standard deviation from two independent experiments.
