In-situ hybridisation to messenger RNA in Heterodera glycines
A method is presented for in-situ hybridization to mRNA in second-stage juveniles (J2) of the soybean cyst nematode Heterodera glycines. The protocol was developed using a digoxigenin-labeled RNA probe transcribed from cDNA of a cellulase gene that was known to be expressed in the subventral esophageal glands of H. glycines. Formaldehyde-fixed J2 were cut into sections with a vibrating razor blade to make the inside of the nematodes accessible for probing. These nematode fragments then were hybridized in suspension with riboprobe, and labeled with an alkaline phosphatase-conjugated antibody to digoxigenin. Staining with nitroblue tetrazolium and bromo-chloro-indolyl phosphate revealed a highly specific hybridization signal to mRNA within the cytoplasm of the subventral gland cells, using this specific antisense probe. This in-situ hybridization protocol will be useful for the characterization and identification of esophageal gland secretion genes in plant-parasitic nematodes, among other applications. Key words: cellulase gene, digoxigenin RNA probe, esophageal gland, Heterodera glycines, in-situ hybridization, nematode.
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Format: | Article/Letter to editor biblioteca |
Language: | English |
Subjects: | Cellulase gene, Digoxigenin RNA probe, Esophageal gland, Heterodera glycines, In-situ hybridization, Nematode, |
Online Access: | https://research.wur.nl/en/publications/in-situ-hybridisation-to-messenger-rna-in-heterodera-glycines |
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dig-wur-nl-wurpubs-602062024-09-23 de Boer, J.M. Yitang Yan Smant, G. Davis, E.L. Baum, T.J. Article/Letter to editor Journal of Nematology 30 (1998) 3 ISSN: 0022-300X In-situ hybridisation to messenger RNA in Heterodera glycines 1998 A method is presented for in-situ hybridization to mRNA in second-stage juveniles (J2) of the soybean cyst nematode Heterodera glycines. The protocol was developed using a digoxigenin-labeled RNA probe transcribed from cDNA of a cellulase gene that was known to be expressed in the subventral esophageal glands of H. glycines. Formaldehyde-fixed J2 were cut into sections with a vibrating razor blade to make the inside of the nematodes accessible for probing. These nematode fragments then were hybridized in suspension with riboprobe, and labeled with an alkaline phosphatase-conjugated antibody to digoxigenin. Staining with nitroblue tetrazolium and bromo-chloro-indolyl phosphate revealed a highly specific hybridization signal to mRNA within the cytoplasm of the subventral gland cells, using this specific antisense probe. This in-situ hybridization protocol will be useful for the characterization and identification of esophageal gland secretion genes in plant-parasitic nematodes, among other applications. Key words: cellulase gene, digoxigenin RNA probe, esophageal gland, Heterodera glycines, in-situ hybridization, nematode. en application/pdf https://research.wur.nl/en/publications/in-situ-hybridisation-to-messenger-rna-in-heterodera-glycines https://edepot.wur.nl/669879 Cellulase gene Digoxigenin RNA probe Esophageal gland Heterodera glycines In-situ hybridization Nematode (c) publisher Wageningen University & Research |
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Cellulase gene Digoxigenin RNA probe Esophageal gland Heterodera glycines In-situ hybridization Nematode Cellulase gene Digoxigenin RNA probe Esophageal gland Heterodera glycines In-situ hybridization Nematode |
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Cellulase gene Digoxigenin RNA probe Esophageal gland Heterodera glycines In-situ hybridization Nematode Cellulase gene Digoxigenin RNA probe Esophageal gland Heterodera glycines In-situ hybridization Nematode de Boer, J.M. Yitang Yan Smant, G. Davis, E.L. Baum, T.J. In-situ hybridisation to messenger RNA in Heterodera glycines |
description |
A method is presented for in-situ hybridization to mRNA in second-stage juveniles (J2) of the soybean cyst nematode Heterodera glycines. The protocol was developed using a digoxigenin-labeled RNA probe transcribed from cDNA of a cellulase gene that was known to be expressed in the subventral esophageal glands of H. glycines. Formaldehyde-fixed J2 were cut into sections with a vibrating razor blade to make the inside of the nematodes accessible for probing. These nematode fragments then were hybridized in suspension with riboprobe, and labeled with an alkaline phosphatase-conjugated antibody to digoxigenin. Staining with nitroblue tetrazolium and bromo-chloro-indolyl phosphate revealed a highly specific hybridization signal to mRNA within the cytoplasm of the subventral gland cells, using this specific antisense probe. This in-situ hybridization protocol will be useful for the characterization and identification of esophageal gland secretion genes in plant-parasitic nematodes, among other applications. Key words: cellulase gene, digoxigenin RNA probe, esophageal gland, Heterodera glycines, in-situ hybridization, nematode. |
format |
Article/Letter to editor |
topic_facet |
Cellulase gene Digoxigenin RNA probe Esophageal gland Heterodera glycines In-situ hybridization Nematode |
author |
de Boer, J.M. Yitang Yan Smant, G. Davis, E.L. Baum, T.J. |
author_facet |
de Boer, J.M. Yitang Yan Smant, G. Davis, E.L. Baum, T.J. |
author_sort |
de Boer, J.M. |
title |
In-situ hybridisation to messenger RNA in Heterodera glycines |
title_short |
In-situ hybridisation to messenger RNA in Heterodera glycines |
title_full |
In-situ hybridisation to messenger RNA in Heterodera glycines |
title_fullStr |
In-situ hybridisation to messenger RNA in Heterodera glycines |
title_full_unstemmed |
In-situ hybridisation to messenger RNA in Heterodera glycines |
title_sort |
in-situ hybridisation to messenger rna in heterodera glycines |
url |
https://research.wur.nl/en/publications/in-situ-hybridisation-to-messenger-rna-in-heterodera-glycines |
work_keys_str_mv |
AT deboerjm insituhybridisationtomessengerrnainheteroderaglycines AT yitangyan insituhybridisationtomessengerrnainheteroderaglycines AT smantg insituhybridisationtomessengerrnainheteroderaglycines AT davisel insituhybridisationtomessengerrnainheteroderaglycines AT baumtj insituhybridisationtomessengerrnainheteroderaglycines |
_version_ |
1816164461208141824 |