Inu uk f1

We mixed Cy3-miD2861 with one sODN of four different sequences (the passenger of miR-2861, matured miR-2861, Cy3-miD2861 and cDNA of HDAC5 mRNA at the miR-2861 binding site) at a 1:1 ratio (100 pmol each in 50 μl) at room temperature. For ex vivo hybridization, we heated the mixture at 65 °C for 5 min, then slowly cooled it on a thermocycler (at a rate of 1° drop per minute) to 20 °C, where it was maintained for 30 min. We resolved the hybrids in agarose gel (1.5%) using gel electrophoresis, and obtained photographs at 495 nm/521 nm (excitation and emission spectrum peak wavelengths) using an Imager FluorChem Q (Alpha Innotech, CA). To test binding ability in vivo, we transfected Cy2-miD2861 (30 pmol) to PC-12 cells in a 35 mm (dia) culture dish on a microscope stage incubator chamber with humidified air comtaining 5% CO2, at 37 °C (model INU-UK-F1; Tokai Hit, Shizuoka, Japan) as described [29 (link)]. We washed and changed DNA-free medium 3 h later, then transfected Cy3-sODNhdac5 to both plates. We acquired live cell images at 18 and 42 min and 2 h after Cy3-sODNhdac5 using automatic time-lapse photography with constant exposure time and image gain (CellSense Imaging Software, Olumpus). Representative cell images were processed using Adobe Photoshop CS2 (Adobe Systems, San Jose, CA).