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                                        Nanoprobes protocol:

                                        RNA Labeling with Nanogold®

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                                        This procedure couples the 3' end of RNA to Nanogold®. It is based upon an oxidation of the ribose cis diols (2' and 3' position) with periodate to aldehydes, reacting this with Monoamino-Nanogold® to form the Schiff's base, then reducing this to form a stable bond.

                                         

                                        [Reaction diagram (4k)]

                                         

                                        Protocol:

                                        1. All steps at 4°C; use DEPC (diethyl pyrocarbonate, 500 microliters/liter, mix for 30 minutes at room temperature, autoclave) treated solutionsif possible. This is to remove RNAse. Wear gloves.
                                        2. Periodate treatment:

                                          For example, take 500 pmoles of RNA, add NaIO4 (1,000 nmoles, freshly prepared). Buffer is 100 mM PIPES, pH 7, 20 microliters and100 microliters water. Incubate at 4°C for 90 minutes. Quench with 2 microliters of sterile glycerol for 5 minutes. Add water to a volumeof 500 microliters, apply to NAP-S column (Pharmacia, GH25 material), elute with 1 mL ofwater.Ethanol precipitate the RNA: ad 2.5 volumes of -20°C chilled ethanol (~2.5 mL), 1/15 volume of 3M NaOAc, pH 5.0 (~70 microliters). Keep at -20°C overnight or -80°C for at least 30 minutes. Spin at 13,000 rpm for 4 minutes. Wash pellet carefully with 100 microliters of -20°C, 70 % EtOH. Spin again for 5minutes, dry pellet in Speedvac. Redissolve in 60 microliters of water.

                                        3. Gold Coupling:

                                          Take 30 nmoles of Monoamino-Nanogold® up to 100 microliters in anhydrous DMSO. Mix: Au-DMSO (16.7 microliters, 10-fold excess); 100 mM PIPES (30 microliters); oxidized RNA (60 microliters); and water (43 microliters). Incubate at 4°C for 60 minutes.

                                        4. Reduction:

                                          Add 1.5 microliters of fresh 20 mg/mL borane tert - butylamine complex (stable for ~15 min). incubate at 4°C for 30 minutes. Stop reaction with 2 microliters of acetone. After 3 minutes at 4°C, apply to a S200 column (RNAse free; DEPC treated): 1 x 75 cm in 0.1 M Tris-HCl, pH 7. Recovered RNA ~80 %, >50 % gold labeled.

                                         

                                        Thanks to W.-D. Hardt for sharing these reaction details.

                                         

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