In this study, pre-miR-664a, pre-miR-664a-U1A, and pre-miRNA-U1A control (non-targeting pre-miRNA containing the U1A recognition sequence) were prepared by in vitro transcription. To generate DNA templates for transcription, primer extension was performed using 2 µM of each primer (shown in Table S1 ) in a reaction mixture containing KOD Dash DNA polymerase (TOYOBO, Osaka, Japan). The resulting template DNA was precipitated with 2-propanol. T7 RNA polymerase was prepared as described previously21 (link). The transcription reaction was carried out at 37 °C for 4 h in a reaction mixture containing 40 mM Tris–HCl (pH 8.0), 24 mM MgCl2, 5 mM dithiothreitol, 10 mM guanosine monophosphate, 2 mM of each NTP, 1.8 U/mL inorganic pyrophosphatase (Sigma, St. Louis, MO, USA), 26.2 µg/mL purified T7 RNA polymerase, and 10 µg/mL DNA template. The pre-miRNA transcripts were purified using an 8% denaturing polyacrylamide gel. All pre-miRNAs were renatured by incubating for 1 min at 85 °C, followed by slow cooling to 4 °C.
The FAM-labeled RNA (RNA-FAM) was purchased from Hokkaido System Science (Hokkaido, Japan). The RNA-FAM sequence was as follows: 5′-GAU UAU GUC CGG UUA UGU ACA UUG CAC UCC G UA CAU AAC CGG ACA UAA UCdT dT-FAM-3′ (the U1A binding sequence is underlined).
The FAM-labeled RNA (RNA-FAM) was purchased from Hokkaido System Science (Hokkaido, Japan). The RNA-FAM sequence was as follows: 5′-GAU UAU GUC CGG UUA UGU A
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