Figure 4. In vitro annealing kinetics between miR-224 and TCF21 3′UTR variants.

(A) (Left panel) TCF21 3′-UTR variants were generated by in vitro transcription (IVT) and incubated with excess over ³²P-labeled miR-224 for various time points, followed by autoradiography detection. Band intensities indicate relative amounts of the 3′-UTR variant:miRNA complexes formed over indicated times. (Right panel) IVT 3′-UTR variants were also incubated with excess over ³²P-labeled miR-224_SNP, resulting in a seed mismatch with the C variant and a seed match with the G variant. (B) Band intensities of 3′-UTR:miRNA complexes formed using ³²P-labeled miR-224 or ³²P-labeled miR-224_SNP were detected by PhosphorImager and to quantify the percentage of complex signals ImageQuant-Software was used to determine relative to whole lane signal. Values represent mean ± SEM from three independent experiments. (C) Calculation of second-order rate constants for individual mRNA:miRNA complexes was performed as previously described [54]. n.d., complex formation was too slow to derive a rate constant.