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Selective binding of nucleotide probes by eosinophilic cationic protein during in situ hybridisation
Richard D Ellis1, Paul Ashwood, Jonathan J Powell
1Gastrointestinal Laboratory, The Rayne Institute, St. Thomas' Hospital, London, SE1 7EH, UK.
During in situ hybridisation on frozen and paraffin-embedded sections of bowel for IkappaB alpha, oligodeoxyribonucleotide probes were found to bind more avidly to eosinophils than target mRNA. This binding could not be obviated using strategies previously employed to block either binding of long DNA probes (200-mers) to eosinophils in bone marrow smears, or of riboprobes to eosinophils in sections of bowel, without removing specific hybridisation of probes. That this binding could arise through interaction of anionic oligodeoxyribonucleotides with eosinophil cationic protein, which has an unusually high pI, and is abundant in cytoplasmic granules of eosinophils, was demonstrated in vitro using real-time biomolecular interaction analysis with a BiacoreX instrument. Finally, a relationship between probe hydrophobicity, measured by reverse phase ion-pair high performance liquid chromatography, and in situ binding of individual probes to eosinophils was demonstrated. Effective tissue penetration by hydrophobic probes and subsequent strong probe-eosinophilic cationic protein interactions therefore may confound the interpretation of in situ hybridisation performed with oligonucleotide probes in eosinophil-containing tissues, such as bowel and nasal polyps.
During in situ hybridisation on frozen and paraffin-embedded sections of bowel for IkappaB alpha, oligodeoxyribonucleotide probes were found to bind more avidly to eosinophils than target mRNA. This binding could not be obviated using strategies previously employed to block either binding of long DNA probes (200-mers) to eosinophils in bone marrow smears, or of riboprobes to eosinophils in sections of bowel, without removing specific hybridisation of probes. That this binding could arise through interaction of anionic oligodeoxyribonucleotides with eosinophil cationic protein, which has an unusually high pI, and is abundant in cytoplasmic granules of eosinophils, was demonstrated in vitro using real-time biomolecular interaction analysis with a BiacoreX instrument. Finally, a relationship between probe hydrophobicity, measured by reverse phase ion-pair high performance liquid chromatography, and in situ binding of individual probes to eosinophils was demonstrated. Effective tissue penetration by hydrophobic probes and subsequent strong probe-eosinophilic cationic protein interactions therefore may confound the interpretation of in situ hybridisation performed with oligonucleotide probes in eosinophil-containing tissues, such as bowel and nasal polyps.