浅谈northern blot-2.ppt

Northern Blot for microRNA 营养与食品卫生学教研室 路慧敏 Biogenesis and Action of miRNAs Conventional Detecting Techniques Northern Blot Quantitative Real time-RT-PCR History Northern Blot Strategy Harvest tissues/cells and extract RNA Electrophoresis and blotting Design label probe of known sequence Hybridization Visualize quantify signal miRNA extraction mirVana? miRNA Isolation Kit (Ambion, Inc) TRIzol total RNA isolation miRNA extraction mirVana? miRNA Isolation Kit (Ambion, Inc) miRNA extraction TRIzol RNA isolation miRNA extraction miRNA extraction OD (optical density ) OD260 OD280 OD230 OD260/OD280: 1.8-2.0 OD260/230 2.0 Northern Blot Strategy Harvest tissues/cells and extract RNA Electrophoresis and blotting Design label probe of known sequence Hybridization Visualize quantify signal Electrophoresis and blotting Preparation of equipment and reagents Soak the gel tanks, combs etc in 0.2 M NaOH for 15 minutes, and then in DEPC-treated water for 15 min. All reagents should be newly opened or use specific for RNA and constructed with DEPC-treated water. Electrophoresis and blotting Electrophoresis and blotting Agarose Gel Electrophoresis and blotting 10 ug RNA + 2X loading buffer Incubated at 65℃ for 10min. On ice for 1min. Run 100 V for 19min. Running buffer (1X MOPS) 1. Set up gel apparatus 2. Make up 15% Denaturing Gel 3. Allow gel to polymerize for 1 hour 4. Assemble gel apparatus and add the running buffer (0.5X TBE) 5. Clean out wells with running buffer- make sure there are NO leaks!!! 6. Pre-run the gel at 180 volts for 30 min 7. Rinse wells right before loading sample 8. Load samples and run at 180 volts until the dye reaches the bottom of the gel Vacuum transfer (Agarose Gel) Semi-Dry transfer (Acrylamide Gel) Vacuum transfer (Agarose Gel) Semi-Dry transfer (Acrylamide Gel) Wash blot in 2X SSC or 0.5X TBE to remove any traces of the gel Place wet membrane on a wet sheet of filter paper and UV Crosslink at optimal setting Stor