Liquid phase fluorescence in situ RT-PCR analysis for gene expression analysis in woody stems.
Identifieur interne : 004141 ( Main/Exploration ); précédent : 004140; suivant : 004142Liquid phase fluorescence in situ RT-PCR analysis for gene expression analysis in woody stems.
Auteurs : M. Gray-Mitsumune [Suède] ; H. Abe ; J. Takahashi ; B. Sundberg ; E J MellerowiczSource :
- Plant biology (Stuttgart, Germany) [ 1435-8603 ]
Descripteurs français
- KwdFr :
- ADN des plantes (génétique), ARN des plantes (génétique), ARN des plantes (isolement et purification), Amorces ADN (génétique), Analyse de profil d'expression de gènes (MeSH), Bois (MeSH), Gènes de plante (MeSH), Hybridation fluorescente in situ (MeSH), Microscopie confocale (MeSH), Populus (enzymologie), Populus (génétique), RT-PCR (MeSH), Ribulose bisphosphate carboxylase (génétique), Séquence nucléotidique (MeSH), Tiges de plante (génétique).
- MESH :
- enzymologie : Populus.
- génétique : ADN des plantes, ARN des plantes, Amorces ADN, Populus, Ribulose bisphosphate carboxylase, Tiges de plante.
- isolement et purification : ARN des plantes.
- Analyse de profil d'expression de gènes, Bois, Gènes de plante, Hybridation fluorescente in situ, Microscopie confocale, RT-PCR, Séquence nucléotidique.
English descriptors
- KwdEn :
- Base Sequence (MeSH), DNA Primers (genetics), DNA, Plant (genetics), Gene Expression Profiling (MeSH), Genes, Plant (MeSH), In Situ Hybridization, Fluorescence (MeSH), Microscopy, Confocal (MeSH), Plant Stems (genetics), Populus (enzymology), Populus (genetics), RNA, Plant (genetics), RNA, Plant (isolation & purification), Reverse Transcriptase Polymerase Chain Reaction (MeSH), Ribulose-Bisphosphate Carboxylase (genetics), Wood (MeSH).
- MESH :
- chemical , genetics : DNA Primers, DNA, Plant, RNA, Plant, Ribulose-Bisphosphate Carboxylase.
- enzymology : Populus.
- genetics : Plant Stems, Populus.
- chemical , isolation & purification : RNA, Plant.
- Base Sequence, Gene Expression Profiling, Genes, Plant, In Situ Hybridization, Fluorescence, Microscopy, Confocal, Reverse Transcriptase Polymerase Chain Reaction, Wood.
Abstract
We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen ( Populus tremula L. x tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.
DOI: 10.1055/s-2003-44747
PubMed: 15095134
Affiliations:
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Abe</name></author><author><name sortKey="Takahashi, J" sort="Takahashi, J" uniqKey="Takahashi J" first="J" last="Takahashi">J. Takahashi</name></author><author><name sortKey="Sundberg, B" sort="Sundberg, B" uniqKey="Sundberg B" first="B" last="Sundberg">B. Sundberg</name></author><author><name sortKey="Mellerowicz, E J" sort="Mellerowicz, E J" uniqKey="Mellerowicz E" first="E J" last="Mellerowicz">E J Mellerowicz</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2004">2004 Jan-Feb</date><idno type="RBID">pubmed:15095134</idno><idno type="pmid">15095134</idno><idno type="doi">10.1055/s-2003-44747</idno><idno type="wicri:Area/Main/Corpus">004284</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Corpus" wicri:corpus="PubMed">004284</idno><idno type="wicri:Area/Main/Curation">004284</idno><idno type="wicri:explorRef" wicri:stream="Main" wicri:step="Curation">004284</idno><idno type="wicri:Area/Main/Exploration">004284</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Liquid phase fluorescence in situ RT-PCR analysis for gene expression analysis in woody stems.</title><author><name sortKey="Gray Mitsumune, M" sort="Gray Mitsumune, M" uniqKey="Gray Mitsumune M" first="M" last="Gray-Mitsumune">M. Gray-Mitsumune</name><affiliation wicri:level="1"><nlm:affiliation>Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå 901 83, Sweden.</nlm:affiliation><country xml:lang="fr">Suède</country><wicri:regionArea>Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå 901 83</wicri:regionArea><wicri:noRegion>Umeå 901 83</wicri:noRegion></affiliation></author><author><name sortKey="Abe, H" sort="Abe, H" uniqKey="Abe H" first="H" last="Abe">H. Abe</name></author><author><name sortKey="Takahashi, J" sort="Takahashi, J" uniqKey="Takahashi J" first="J" last="Takahashi">J. Takahashi</name></author><author><name sortKey="Sundberg, B" sort="Sundberg, B" uniqKey="Sundberg B" first="B" last="Sundberg">B. Sundberg</name></author><author><name sortKey="Mellerowicz, E J" sort="Mellerowicz, E J" uniqKey="Mellerowicz E" first="E J" last="Mellerowicz">E J Mellerowicz</name></author></analytic><series><title level="j">Plant biology (Stuttgart, Germany)</title><idno type="ISSN">1435-8603</idno></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Base Sequence (MeSH)</term><term>DNA Primers (genetics)</term><term>DNA, Plant (genetics)</term><term>Gene Expression Profiling (MeSH)</term><term>Genes, Plant (MeSH)</term><term>In Situ Hybridization, Fluorescence (MeSH)</term><term>Microscopy, Confocal (MeSH)</term><term>Plant Stems (genetics)</term><term>Populus (enzymology)</term><term>Populus (genetics)</term><term>RNA, Plant (genetics)</term><term>RNA, Plant (isolation & purification)</term><term>Reverse Transcriptase Polymerase Chain Reaction (MeSH)</term><term>Ribulose-Bisphosphate Carboxylase (genetics)</term><term>Wood (MeSH)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN des plantes (génétique)</term><term>ARN des plantes (génétique)</term><term>ARN des plantes (isolement et purification)</term><term>Amorces ADN (génétique)</term><term>Analyse de profil d'expression de gènes (MeSH)</term><term>Bois (MeSH)</term><term>Gènes de plante (MeSH)</term><term>Hybridation fluorescente in situ (MeSH)</term><term>Microscopie confocale (MeSH)</term><term>Populus (enzymologie)</term><term>Populus (génétique)</term><term>RT-PCR (MeSH)</term><term>Ribulose bisphosphate carboxylase (génétique)</term><term>Séquence nucléotidique (MeSH)</term><term>Tiges de plante (génétique)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA Primers</term><term>DNA, Plant</term><term>RNA, Plant</term><term>Ribulose-Bisphosphate Carboxylase</term></keywords><keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Populus</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Populus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Plant Stems</term><term>Populus</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ADN des plantes</term><term>ARN des plantes</term><term>Amorces ADN</term><term>Populus</term><term>Ribulose bisphosphate carboxylase</term><term>Tiges de plante</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>RNA, Plant</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>ARN des plantes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Base Sequence</term><term>Gene Expression Profiling</term><term>Genes, Plant</term><term>In Situ Hybridization, Fluorescence</term><term>Microscopy, Confocal</term><term>Reverse Transcriptase Polymerase Chain Reaction</term><term>Wood</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Analyse de profil d'expression de gènes</term><term>Bois</term><term>Gènes de plante</term><term>Hybridation fluorescente in situ</term><term>Microscopie confocale</term><term>RT-PCR</term><term>Séquence nucléotidique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen ( Populus tremula L. x tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15095134</PMID><DateCompleted><Year>2004</Year><Month>05</Month><Day>19</Day></DateCompleted><DateRevised><Year>2011</Year><Month>11</Month><Day>17</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">1435-8603</ISSN><JournalIssue CitedMedium="Print"><Volume>6</Volume><Issue>1</Issue><PubDate><MedlineDate>2004 Jan-Feb</MedlineDate></PubDate></JournalIssue><Title>Plant biology (Stuttgart, Germany)</Title><ISOAbbreviation>Plant Biol (Stuttg)</ISOAbbreviation></Journal><ArticleTitle>Liquid phase fluorescence in situ RT-PCR analysis for gene expression analysis in woody stems.</ArticleTitle><Pagination><MedlinePgn>47-54</MedlinePgn></Pagination><Abstract><AbstractText>We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen ( Populus tremula L. x tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Gray-Mitsumune</LastName><ForeName>M</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå 901 83, Sweden.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Abe</LastName><ForeName>H</ForeName><Initials>H</Initials></Author><Author ValidYN="Y"><LastName>Takahashi</LastName><ForeName>J</ForeName><Initials>J</Initials></Author><Author ValidYN="Y"><LastName>Sundberg</LastName><ForeName>B</ForeName><Initials>B</Initials></Author><Author ValidYN="Y"><LastName>Mellerowicz</LastName><ForeName>E J</ForeName><Initials>EJ</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Plant Biol (Stuttg)</MedlineTA><NlmUniqueID>101148926</NlmUniqueID><ISSNLinking>1435-8603</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D017931">DNA Primers</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D018744">DNA, Plant</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D018749">RNA, Plant</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 4.1.1.39</RegistryNumber><NameOfSubstance UI="D012273">Ribulose-Bisphosphate Carboxylase</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D001483" MajorTopicYN="N">Base Sequence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017931" MajorTopicYN="N">DNA Primers</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D018744" MajorTopicYN="N">DNA, Plant</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D020869" MajorTopicYN="N">Gene Expression Profiling</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017343" MajorTopicYN="N">Genes, Plant</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017404" MajorTopicYN="N">In Situ Hybridization, Fluorescence</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D018613" MajorTopicYN="N">Microscopy, Confocal</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D018547" MajorTopicYN="N">Plant Stems</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D032107" MajorTopicYN="N">Populus</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="N">enzymology</QualifierName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D018749" MajorTopicYN="N">RNA, Plant</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000302" MajorTopicYN="N">isolation & purification</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D020133" MajorTopicYN="N">Reverse Transcriptase Polymerase Chain Reaction</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D012273" MajorTopicYN="N">Ribulose-Bisphosphate Carboxylase</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014934" MajorTopicYN="N">Wood</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2004</Year><Month>4</Month><Day>20</Day><Hour>5</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2004</Year><Month>5</Month><Day>20</Day><Hour>5</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2004</Year><Month>4</Month><Day>20</Day><Hour>5</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">15095134</ArticleId><ArticleId IdType="doi">10.1055/s-2003-44747</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Suède</li></country></list><tree><noCountry><name sortKey="Abe, H" sort="Abe, H" uniqKey="Abe H" first="H" last="Abe">H. Abe</name><name sortKey="Mellerowicz, E J" sort="Mellerowicz, E J" uniqKey="Mellerowicz E" first="E J" last="Mellerowicz">E J Mellerowicz</name><name sortKey="Sundberg, B" sort="Sundberg, B" uniqKey="Sundberg B" first="B" last="Sundberg">B. Sundberg</name><name sortKey="Takahashi, J" sort="Takahashi, J" uniqKey="Takahashi J" first="J" last="Takahashi">J. Takahashi</name></noCountry><country name="Suède"><noRegion><name sortKey="Gray Mitsumune, M" sort="Gray Mitsumune, M" uniqKey="Gray Mitsumune M" first="M" last="Gray-Mitsumune">M. Gray-Mitsumune</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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