Total RNA was isolated from miRNeasy kit (Qiagen) and reversely transcribed by miScript PCR starter kit. Qiagen's miScript PCR system was used to detect miR-200c and miR-15a transcription. MiR-200c was cloned into pLVX-puro (Clonetech) for inducible expression. Cells were exposed to doxycline at final concentration of 4 μg/ml to induce miR-200c expression.
In vitro microRNA binding assay was performed as described (23 (link)). In short, Bluescript plasmid containing the Pin1 3' UTR was used as template in PCR. Forward primer located at the vector. Reverse primer is from the DNA sequence of miR-200c. The parameters for the PCR reaction were: one cycle at 95°C for 5 min; 35 cycles at 95°C for 1 min, 37°C for 1 min, 72°C for 1 min; and a final elongation step at 72°C for 10 min. The PCR products were then visualized with a 1.5% agarose gel stained with ethidium bromide.
For the reporter assay of Pin1 3'UTR by miR-200c, wildtype and mutant Pin1 3'UTR was cloned into a luciferase construct psiCHECK2 (Promega). Cells were harvested and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega).
In vitro microRNA binding assay was performed as described (23 (link)). In short, Bluescript plasmid containing the Pin1 3' UTR was used as template in PCR. Forward primer located at the vector. Reverse primer is from the DNA sequence of miR-200c. The parameters for the PCR reaction were: one cycle at 95°C for 5 min; 35 cycles at 95°C for 1 min, 37°C for 1 min, 72°C for 1 min; and a final elongation step at 72°C for 10 min. The PCR products were then visualized with a 1.5% agarose gel stained with ethidium bromide.
For the reporter assay of Pin1 3'UTR by miR-200c, wildtype and mutant Pin1 3'UTR was cloned into a luciferase construct psiCHECK2 (Promega). Cells were harvested and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega).
