Nonradioactive Northern blotting with biotinylated and digoxigenin-labeled RNA probes
| Autor: | Hans-Joachim Hoeltke, Nathalie Chiasson, Arnold H. Greenberg, Karryn L. Garrett, Jon C. Meltzer, Veronica Sanders, Paul C. Grimm, Dwight M. Nance |
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| Rok vydání: | 1998 |
| Předmět: |
chemistry.chemical_classification
Male Clinical Biochemistry RNA Biotin RNA Probes Blotting Northern Biochemistry Stripping (fiber) Analytical Chemistry Rats Rats Sprague-Dawley chemistry.chemical_compound Membrane chemistry Biotinylation Digoxigenin Animals Nucleotide Indicators and Reagents Northern blot |
| Zdroj: | Electrophoresis. 19(8-9) |
| ISSN: | 0173-0835 |
| Popis: | The application of nonradioactive RNA probes for Northern blotting offers the advantage of a rapid turn-around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)-labeled RNA probes from nylon membranes. One protocol utilizes a phosphate-buffered formamide stripping solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripping solution. This latter method was developed by Ambion Inc. and is called Strip-EZ™. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin-labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called ChemLink™, that quickly and conveniently labels RNA for use in Northern blotting. |
| Databáze: | OpenAIRE |
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