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5. Spin the tube at 13,000 rpm for 2 min. Transfer the aqueous phase to a Wizard SV minicolumn (included in Wizard Plus SV Minipreps DNA Purification System, Promega, Cat# A1460), place the minicolumn in a microcentrifuge tube, and spin at 13,000 rpm for 2 min. 6. Transfer the aqueous phase (∼450 μL) to a new microcentrifuge tube. 5 μL of glycogen and 1 mL of –20 ºC ethanol. Invert the tube several times to mix well. Incubate at –20 ºC for 1 h to overnight. 7. Spin at 13,000 rpm for 5 min at 4 ºC.
When performing radioactive experiments, always take care to avoid contaminating the environment, and protect yourself and people around you from exposure to the radioactive materials. 7. To minimize RNase contamination, all solutions and buffers used in these experiments should be made with DEPC-treated water. Always wear gloves, and use RNase-free tips and reagents designated to the RNA bench. 8. When preparing radiolabeled dsRNA or pre-miRNA substrates, it is best to verify the quality of the radiolabeled probes by running 1 μL of probe on a denaturing PAGE prior to performing the actual assays.
Nature 418, 244–251. 2. Bartel, D. P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116,281–297. 3. , Kolb, F. , and Pillai, R. S. (2005). Posttranscriptional gene silencing by siRNAs and miRNAs. Curr. Opin. Struct. Biol. 15, 331–341. 4. Sontheimer, E. , and Carthew, R. W. (2005). Silence from within: Endogenous siRNAs and miRNAs. Cell 122, 9–12. 5. , and Slack, F. J. (2006). Oncomirs—MicroRNAs with a role in cancer. Nat. Rev. Cancer 6, 259–269. 6. Plasterk, R. H. (2006).