In Situ Detection of Messenger RNA Using Digoxigenin-Labeled Oligonucleotides and Rolling Circle Amplification
| Autor: | John H. Leamon, Yi Zhou, Tod Strugnell, Stefan Hamann, Matthew W. Christian, Margaret Calciano, Paul M. Lizardi |
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| Rok vydání: | 2001 |
| Předmět: |
In situ
Molecular Sequence Data Clinical Biochemistry In situ hybridization Biology Sensitivity and Specificity Cell Line Oligodeoxyribonucleotides Antisense Pathology and Forensic Medicine chemistry.chemical_compound Image Processing Computer-Assisted Animals Digoxigenin RNA Messenger Molecular Biology In Situ Hybridization In Situ Hybridization Fluorescence Microscopy Messenger RNA Base Sequence Oligonucleotide Hybridization probe RNA Molecular biology Actins Rats Liver chemistry Rolling circle replication Immunoglobulin G Biophysics 5' Untranslated Regions DNA Probes Oligonucleotide Probes |
| Zdroj: | Experimental and Molecular Pathology. 70:281-288 |
| ISSN: | 0014-4800 |
| DOI: | 10.1006/exmp.2001.2365 |
| Popis: | The detection of specific RNA molecules in situ is routinely performed using haptenated probes, which are detected by either enzymatic amplification or direct fluorescence. A drawback of fluorescence labeling has been the reduced sensitivity relative to that of methods that use enzymes as signal generators. Reliable fluorescence detection methods often require the use of multiple oligonucleotide probes for each gene target. Here, we demonstrate that single haptenated DNA probes specific for actin mRNA may be detected in situ using antibody-coupled rolling circle amplification (immuno-RCA). This fluorescence-based detection method offers remarkable sensitivity due to the use of signal amplification and yet retains the ability to count hybridization signals as discrete objects. We demonstrate the detection of actin-specific immuno-RCA signals in the cytoplasm and use 3D image deconvolution of multiple z axis sections to show that there are hundreds of signals per cell. With some modifications, this method may be adaptable to the simultaneous detection of several RNA species, including low-copy-number mRNA. |
| Databáze: | OpenAIRE |
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