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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Bibliographic Details
Main Authors: de Boer, J.M., Yitang Yan, Smant, G., Davis, E.L., Baum, T.J.
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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spelling 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
institution WUR NL
collection DSpace
country Países bajos
countrycode NL
component Bibliográfico
access En linea
databasecode dig-wur-nl
tag biblioteca
region Europa del Oeste
libraryname WUR Library Netherlands
language English
topic 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
spellingShingle 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
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