RNA Labeling with Nanogold®

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 glycine 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 60°C for 4 minutes.

4. Reduction:

   Add 1.5 microliters of fresh 20 mg/mL borane tert - butylamine complex (stable for ~15 min). incubate at 30°C for 4 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.

For more information contact:

Email us at tech@nanoprobes.com.

Return to catalog: Nanogold® Labeling Reagents

Other Applications:

Product Applications Special report: A very reliable pre-embedding immunogold method. Our 2002 paper: Nickel-NTA-Nanogold Binds his-Tagged Proteins. Our 2001 paper on DNA Nanowires. Our 1999 paper on Gold-Based Autometallography. Special report: In Situ Hybridization with Nanogold®-Streptavidin. Technical note: Nanogold® Staining on Gels. Our 1996 paper on Gold Liposomes. Special report: Nanogold® Labeling of Peptides. Technical note: RNA Labeling with Nanogold®. Our 1991 paper on A New 1.4 nm Gold-Fab' Probe. Our 1994 paper on Methylamine Vanadate (NanoVan) Negative Stain. Our 1990 paper on Conducting Polymer Films as EM Substrates. Research Applications Our 2005 paper: 5 nm Gold-Ni-NTA binds His Tags. Enzymatic Metallography: A Simple New Staining Method Our 2006 paper on Light and Electron Microscopy of Microsporida using Enzyme Metallography. Our 2004 paper on Enzymatic Metallography as a Correlative Light and Electron Microscopic Method. Our 2002 paper on Enzymatic Metallography: A Simple New Staining Method.

Research Applications (continued) Our 2007 paper on Correlative Enzymatic and Gold Probes for Light and Electron Microscopy. Our 1995 paper on Aldehyde Gold Clusters for Molecular Labeling. Our 2000 paper on Gold Cluster Crystals. Larger covalent gold probes: Our 2005 paper on Preparation of Covalent 3nm Gold – Fab’ Probes. Our 1999 paper on A Covalently Linked 10 nm Gold Immunoprobe. Combined fluorescent and gold probes: Our 2003 paper featuring Dextran-Texas Red®-Nanogold® Fluorescent Dyes for Use in Correlative Microscopy and Intravital Imaging. Our 2002 paper on Combined ALEXA-488 and Nanogold Antibody Probes. Our 1999 paper on Combined Cy3 / Nanogold Probes for Immunocytochemistry and In Situ Hybridization. Our 1998 paper on Dual-Labeled Probes for Fluorescence and Electron Microscopy. News: our original 1997 paper on Combined Fluorescent and Gold Immunoprobes. Our 1996 paper on Large Cluster and Combined Fluorescent and Gold Immunoprobes. Our 1994 paper on Combined Fluorescent and Gold Nucleic Acid Probes. Our 2005 paper on High Z Metal Carbonyls for Imaging and Microspectroscopy. Our 2005 paper on In Vivo Vascular Casting.