Protocol: Analysis of brain mRNA by reverse transcription-polymerase chain reaction and hybridization with digoxigenin-labeled DNA probe

The interleukin-1 family of polypeptides (IL-1 alpha , IL-1 beta and IL-1 receptor antagonist (RA)) induces various centrally mediated host defense responses to infectious pathogens [3]. Considerable interest has focussed on IL-1 as a mediator in disease and in the production of systemic acute phase...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Brain research. Brain research protocols 1997-05, Vol.1 (2), p.145-151
Hauptverfasser: Gabellec, Marie-Madeleine, Griffais, Remy, Fillion, Gilles, Haour, France
Format: Artikel
Sprache:eng
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
container_end_page 151
container_issue 2
container_start_page 145
container_title Brain research. Brain research protocols
container_volume 1
creator Gabellec, Marie-Madeleine
Griffais, Remy
Fillion, Gilles
Haour, France
description The interleukin-1 family of polypeptides (IL-1 alpha , IL-1 beta and IL-1 receptor antagonist (RA)) induces various centrally mediated host defense responses to infectious pathogens [3]. Considerable interest has focussed on IL-1 as a mediator in disease and in the production of systemic acute phase responses. We have recently studied the effects of a peripheral stimulation by intraperitoneal (i.p.) administration of lipoposaccharide (LPS) on the mRNAs expressions of IL-1 ( alpha , beta , RA) and their receptors (IL-1 receptor type I and type II (IL-1R1, IL-1R2)) in the central nervous system (CNS) [5, 6]. The levels of these expressions being very low in the CNS, the reverse transcription-polymerase chain reaction (RT-PCR) techniques are required for these studies. RT-PCR is a developed method of identifying mRNAs in very small amount of nucleic acid. We have previously developed a method to choose specific PCR primers [9]. The detection of specific PCR products is extremely important. Since amplifications with these specific PCR primers can be achieved under the same conditions (buffers and temperatures) reliable results can be obtained. Characterization of a PCR product requires the use of a specific DNA probe that hybridizes to the region of interest. In addition to providing specificity of detection, the use of labeled DNA probes provides increased sensitivity over ethidium bromide staining. We have previously described a method of synthesis of non-radioactive probe labeled with digoxigenin by nested PCR [5, 6, 8]. Moreover the major advantage to the use of non-radioactive label is that it does not have a short half-life and can last for weeks or even months. A quantification of the PCR products can be obtained using a method based on the analysis of photographic negatives of agarose gels. (c) 1997 Elsevier Science B.V. All rights reserved.
doi_str_mv 10.1016/S1385-299X(96)00023-2
format Article
fullrecord <record><control><sourceid>proquest</sourceid><recordid>TN_cdi_proquest_miscellaneous_20485357</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>20485357</sourcerecordid><originalsourceid>FETCH-proquest_miscellaneous_204853573</originalsourceid><addsrcrecordid>eNqNjb1OwzAURj2ARPl5BKQ7IRgMTtK0DVsFRUwIAQNbZTu3zUWOHXxdIDwBj02KEDPTJ51zpE-I40ydZyqbXDxmxayUeVU9n1aTM6VUXsh8R4z-8J7YZ34ZRDlV45H4uo8hBRvcJcy9dj0TQ1iBiZo8tA93czA9RHzDyAgpas82UpcoeNkF17cY9SBss80jars1oH0NTW8i1fSpf8g7pQZqWocPWqMnL5026LCG6-Ghi8Hgodhdacd49LsH4uRm8XR1Kwf7ukFOy5bYonPaY9jwMlfjWVmU0-Lf4TeXyF0t</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>20485357</pqid></control><display><type>article</type><title>Protocol: Analysis of brain mRNA by reverse transcription-polymerase chain reaction and hybridization with digoxigenin-labeled DNA probe</title><source>Alma/SFX Local Collection</source><creator>Gabellec, Marie-Madeleine ; Griffais, Remy ; Fillion, Gilles ; Haour, France</creator><creatorcontrib>Gabellec, Marie-Madeleine ; Griffais, Remy ; Fillion, Gilles ; Haour, France</creatorcontrib><description>The interleukin-1 family of polypeptides (IL-1 alpha , IL-1 beta and IL-1 receptor antagonist (RA)) induces various centrally mediated host defense responses to infectious pathogens [3]. Considerable interest has focussed on IL-1 as a mediator in disease and in the production of systemic acute phase responses. We have recently studied the effects of a peripheral stimulation by intraperitoneal (i.p.) administration of lipoposaccharide (LPS) on the mRNAs expressions of IL-1 ( alpha , beta , RA) and their receptors (IL-1 receptor type I and type II (IL-1R1, IL-1R2)) in the central nervous system (CNS) [5, 6]. The levels of these expressions being very low in the CNS, the reverse transcription-polymerase chain reaction (RT-PCR) techniques are required for these studies. RT-PCR is a developed method of identifying mRNAs in very small amount of nucleic acid. We have previously developed a method to choose specific PCR primers [9]. The detection of specific PCR products is extremely important. Since amplifications with these specific PCR primers can be achieved under the same conditions (buffers and temperatures) reliable results can be obtained. Characterization of a PCR product requires the use of a specific DNA probe that hybridizes to the region of interest. In addition to providing specificity of detection, the use of labeled DNA probes provides increased sensitivity over ethidium bromide staining. We have previously described a method of synthesis of non-radioactive probe labeled with digoxigenin by nested PCR [5, 6, 8]. Moreover the major advantage to the use of non-radioactive label is that it does not have a short half-life and can last for weeks or even months. A quantification of the PCR products can be obtained using a method based on the analysis of photographic negatives of agarose gels. (c) 1997 Elsevier Science B.V. All rights reserved.</description><identifier>ISSN: 1385-299X</identifier><identifier>DOI: 10.1016/S1385-299X(96)00023-2</identifier><language>eng</language><ispartof>Brain research. Brain research protocols, 1997-05, Vol.1 (2), p.145-151</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,781,785,27928,27929</link.rule.ids></links><search><creatorcontrib>Gabellec, Marie-Madeleine</creatorcontrib><creatorcontrib>Griffais, Remy</creatorcontrib><creatorcontrib>Fillion, Gilles</creatorcontrib><creatorcontrib>Haour, France</creatorcontrib><title>Protocol: Analysis of brain mRNA by reverse transcription-polymerase chain reaction and hybridization with digoxigenin-labeled DNA probe</title><title>Brain research. Brain research protocols</title><description>The interleukin-1 family of polypeptides (IL-1 alpha , IL-1 beta and IL-1 receptor antagonist (RA)) induces various centrally mediated host defense responses to infectious pathogens [3]. Considerable interest has focussed on IL-1 as a mediator in disease and in the production of systemic acute phase responses. We have recently studied the effects of a peripheral stimulation by intraperitoneal (i.p.) administration of lipoposaccharide (LPS) on the mRNAs expressions of IL-1 ( alpha , beta , RA) and their receptors (IL-1 receptor type I and type II (IL-1R1, IL-1R2)) in the central nervous system (CNS) [5, 6]. The levels of these expressions being very low in the CNS, the reverse transcription-polymerase chain reaction (RT-PCR) techniques are required for these studies. RT-PCR is a developed method of identifying mRNAs in very small amount of nucleic acid. We have previously developed a method to choose specific PCR primers [9]. The detection of specific PCR products is extremely important. Since amplifications with these specific PCR primers can be achieved under the same conditions (buffers and temperatures) reliable results can be obtained. Characterization of a PCR product requires the use of a specific DNA probe that hybridizes to the region of interest. In addition to providing specificity of detection, the use of labeled DNA probes provides increased sensitivity over ethidium bromide staining. We have previously described a method of synthesis of non-radioactive probe labeled with digoxigenin by nested PCR [5, 6, 8]. Moreover the major advantage to the use of non-radioactive label is that it does not have a short half-life and can last for weeks or even months. A quantification of the PCR products can be obtained using a method based on the analysis of photographic negatives of agarose gels. (c) 1997 Elsevier Science B.V. All rights reserved.</description><issn>1385-299X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1997</creationdate><recordtype>article</recordtype><recordid>eNqNjb1OwzAURj2ARPl5BKQ7IRgMTtK0DVsFRUwIAQNbZTu3zUWOHXxdIDwBj02KEDPTJ51zpE-I40ydZyqbXDxmxayUeVU9n1aTM6VUXsh8R4z-8J7YZ34ZRDlV45H4uo8hBRvcJcy9dj0TQ1iBiZo8tA93czA9RHzDyAgpas82UpcoeNkF17cY9SBss80jars1oH0NTW8i1fSpf8g7pQZqWocPWqMnL5026LCG6-Ghi8Hgodhdacd49LsH4uRm8XR1Kwf7ukFOy5bYonPaY9jwMlfjWVmU0-Lf4TeXyF0t</recordid><startdate>19970501</startdate><enddate>19970501</enddate><creator>Gabellec, Marie-Madeleine</creator><creator>Griffais, Remy</creator><creator>Fillion, Gilles</creator><creator>Haour, France</creator><scope>7TK</scope><scope>7TM</scope></search><sort><creationdate>19970501</creationdate><title>Protocol: Analysis of brain mRNA by reverse transcription-polymerase chain reaction and hybridization with digoxigenin-labeled DNA probe</title><author>Gabellec, Marie-Madeleine ; Griffais, Remy ; Fillion, Gilles ; Haour, France</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-proquest_miscellaneous_204853573</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1997</creationdate><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gabellec, Marie-Madeleine</creatorcontrib><creatorcontrib>Griffais, Remy</creatorcontrib><creatorcontrib>Fillion, Gilles</creatorcontrib><creatorcontrib>Haour, France</creatorcontrib><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><jtitle>Brain research. Brain research protocols</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gabellec, Marie-Madeleine</au><au>Griffais, Remy</au><au>Fillion, Gilles</au><au>Haour, France</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Protocol: Analysis of brain mRNA by reverse transcription-polymerase chain reaction and hybridization with digoxigenin-labeled DNA probe</atitle><jtitle>Brain research. Brain research protocols</jtitle><date>1997-05-01</date><risdate>1997</risdate><volume>1</volume><issue>2</issue><spage>145</spage><epage>151</epage><pages>145-151</pages><issn>1385-299X</issn><abstract>The interleukin-1 family of polypeptides (IL-1 alpha , IL-1 beta and IL-1 receptor antagonist (RA)) induces various centrally mediated host defense responses to infectious pathogens [3]. Considerable interest has focussed on IL-1 as a mediator in disease and in the production of systemic acute phase responses. We have recently studied the effects of a peripheral stimulation by intraperitoneal (i.p.) administration of lipoposaccharide (LPS) on the mRNAs expressions of IL-1 ( alpha , beta , RA) and their receptors (IL-1 receptor type I and type II (IL-1R1, IL-1R2)) in the central nervous system (CNS) [5, 6]. The levels of these expressions being very low in the CNS, the reverse transcription-polymerase chain reaction (RT-PCR) techniques are required for these studies. RT-PCR is a developed method of identifying mRNAs in very small amount of nucleic acid. We have previously developed a method to choose specific PCR primers [9]. The detection of specific PCR products is extremely important. Since amplifications with these specific PCR primers can be achieved under the same conditions (buffers and temperatures) reliable results can be obtained. Characterization of a PCR product requires the use of a specific DNA probe that hybridizes to the region of interest. In addition to providing specificity of detection, the use of labeled DNA probes provides increased sensitivity over ethidium bromide staining. We have previously described a method of synthesis of non-radioactive probe labeled with digoxigenin by nested PCR [5, 6, 8]. Moreover the major advantage to the use of non-radioactive label is that it does not have a short half-life and can last for weeks or even months. A quantification of the PCR products can be obtained using a method based on the analysis of photographic negatives of agarose gels. (c) 1997 Elsevier Science B.V. All rights reserved.</abstract><doi>10.1016/S1385-299X(96)00023-2</doi></addata></record>
fulltext fulltext
identifier ISSN: 1385-299X
ispartof Brain research. Brain research protocols, 1997-05, Vol.1 (2), p.145-151
issn 1385-299X
language eng
recordid cdi_proquest_miscellaneous_20485357
source Alma/SFX Local Collection
title Protocol: Analysis of brain mRNA by reverse transcription-polymerase chain reaction and hybridization with digoxigenin-labeled DNA probe
url https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-17T02%3A33%3A45IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Protocol:%20Analysis%20of%20brain%20mRNA%20by%20reverse%20transcription-polymerase%20chain%20reaction%20and%20hybridization%20with%20digoxigenin-labeled%20DNA%20probe&rft.jtitle=Brain%20research.%20Brain%20research%20protocols&rft.au=Gabellec,%20Marie-Madeleine&rft.date=1997-05-01&rft.volume=1&rft.issue=2&rft.spage=145&rft.epage=151&rft.pages=145-151&rft.issn=1385-299X&rft_id=info:doi/10.1016/S1385-299X(96)00023-2&rft_dat=%3Cproquest%3E20485357%3C/proquest%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=20485357&rft_id=info:pmid/&rfr_iscdi=true