Total RNA Isolation and Quantification of Specific RNAs in Fission Yeast.
Identifieur interne : 000772 ( Main/Exploration ); précédent : 000771; suivant : 000773Total RNA Isolation and Quantification of Specific RNAs in Fission Yeast.
Auteurs : Robert Roth [États-Unis] ; Hiten D. Madhani [États-Unis] ; Jennifer F. Garcia [États-Unis]Source :
- Methods in molecular biology (Clifton, N.J.) [ 1940-6029 ] ; 2018.
Descripteurs français
- KwdFr :
- MESH :
- biosynthèse : ARN fongique.
- génétique : ARN fongique, Schizosaccharomyces.
- métabolisme : Schizosaccharomyces.
- Analyse de séquence d'ARN, Réaction de polymérisation en chaine en temps réel.
English descriptors
- KwdEn :
- MESH :
- chemical , biosynthesis : RNA, Fungal.
- chemical , genetics : RNA, Fungal.
- genetics : Schizosaccharomyces.
- metabolism : Schizosaccharomyces.
- methods : Real-Time Polymerase Chain Reaction, Sequence Analysis, RNA.
Abstract
The fission yeast, Schizosaccharomyces pombe, is an important model organism for investigations of gene regulation. Essential to such studies is the ability to quantify the levels of a specific RNA. We describe a protocol for the isolation and quantification of RNA in S. pombe using reverse-transcription followed by quantitative PCR. In this procedure, the cells are lysed using zirconia beads, then total RNA is selectively isolated away from proteins and DNA using the Trizol reagent. Contaminating DNA is then removed from the RNA by using TURBO DNase, which is easily inactivated and requires no subsequent clean-up step. The RNA is then reverse transcribed into cDNA using random nine-mers and oligo dT primers . Quantitative PCR using SYBR green is then performed to quantify RNA levels. This protocol has been tested on several S. pombe genotypes and generates highly reproducible results.
DOI: 10.1007/978-1-4939-7546-4_6
PubMed: 29423847
Affiliations:
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Garcia</name><affiliation wicri:level="2"><nlm:affiliation>Department of Molecular Biology, Colorado College, Colorado Springs, CO, USA. jennifer.garcia@coloradocollege.edu.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Molecular Biology, Colorado College, Colorado Springs, CO</wicri:regionArea><placeName><region type="state">Colorado</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2018">2018</date><idno type="RBID">pubmed:29423847</idno><idno type="pmid">29423847</idno><idno type="doi">10.1007/978-1-4939-7546-4_6</idno><idno type="wicri:Area/PubMed/Corpus">000998</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000998</idno><idno type="wicri:Area/PubMed/Curation">000998</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">000998</idno><idno type="wicri:Area/PubMed/Checkpoint">000733</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">000733</idno><idno type="wicri:Area/Ncbi/Merge">001D33</idno><idno type="wicri:Area/Ncbi/Curation">001D33</idno><idno type="wicri:Area/Ncbi/Checkpoint">001D33</idno><idno type="wicri:Area/Main/Merge">000775</idno><idno type="wicri:Area/Main/Curation">000772</idno><idno type="wicri:Area/Main/Exploration">000772</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Total RNA Isolation and Quantification of Specific RNAs in Fission Yeast.</title><author><name sortKey="Roth, Robert" sort="Roth, Robert" uniqKey="Roth R" first="Robert" last="Roth">Robert Roth</name><affiliation wicri:level="2"><nlm:affiliation>Department of Molecular Biology, Colorado College, Colorado Springs, CO, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Molecular Biology, Colorado College, Colorado Springs, CO</wicri:regionArea><placeName><region type="state">Colorado</region></placeName></affiliation></author><author><name sortKey="Madhani, Hiten D" sort="Madhani, Hiten D" uniqKey="Madhani H" first="Hiten D" last="Madhani">Hiten D. Madhani</name><affiliation wicri:level="2"><nlm:affiliation>Deptartment of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Deptartment of Biochemistry and Biophysics, University of California, San Francisco, CA</wicri:regionArea><placeName><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Garcia, Jennifer F" sort="Garcia, Jennifer F" uniqKey="Garcia J" first="Jennifer F" last="Garcia">Jennifer F. Garcia</name><affiliation wicri:level="2"><nlm:affiliation>Department of Molecular Biology, Colorado College, Colorado Springs, CO, USA. jennifer.garcia@coloradocollege.edu.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Molecular Biology, Colorado College, Colorado Springs, CO</wicri:regionArea><placeName><region type="state">Colorado</region></placeName></affiliation></author></analytic><series><title level="j">Methods in molecular biology (Clifton, N.J.)</title><idno type="eISSN">1940-6029</idno><imprint><date when="2018" type="published">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>RNA, Fungal (biosynthesis)</term><term>RNA, Fungal (genetics)</term><term>Real-Time Polymerase Chain Reaction (methods)</term><term>Schizosaccharomyces (genetics)</term><term>Schizosaccharomyces (metabolism)</term><term>Sequence Analysis, RNA (methods)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN fongique (biosynthèse)</term><term>ARN fongique (génétique)</term><term>Analyse de séquence d'ARN ()</term><term>Réaction de polymérisation en chaine en temps réel ()</term><term>Schizosaccharomyces (génétique)</term><term>Schizosaccharomyces (métabolisme)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>RNA, Fungal</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>RNA, Fungal</term></keywords><keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>ARN fongique</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Schizosaccharomyces</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ARN fongique</term><term>Schizosaccharomyces</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Schizosaccharomyces</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Real-Time Polymerase Chain Reaction</term><term>Sequence Analysis, RNA</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Schizosaccharomyces</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Analyse de séquence d'ARN</term><term>Réaction de polymérisation en chaine en temps réel</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The fission yeast, Schizosaccharomyces pombe, is an important model organism for investigations of gene regulation. Essential to such studies is the ability to quantify the levels of a specific RNA. We describe a protocol for the isolation and quantification of RNA in S. pombe using reverse-transcription followed by quantitative PCR. In this procedure, the cells are lysed using zirconia beads, then total RNA is selectively isolated away from proteins and DNA using the Trizol reagent. Contaminating DNA is then removed from the RNA by using TURBO DNase, which is easily inactivated and requires no subsequent clean-up step. The RNA is then reverse transcribed into cDNA using random nine-mers and oligo dT primers . Quantitative PCR using SYBR green is then performed to quantify RNA levels. This protocol has been tested on several S. pombe genotypes and generates highly reproducible results.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Californie</li><li>Colorado</li></region></list><tree><country name="États-Unis"><region name="Colorado"><name sortKey="Roth, Robert" sort="Roth, Robert" uniqKey="Roth R" first="Robert" last="Roth">Robert Roth</name></region><name sortKey="Garcia, Jennifer F" sort="Garcia, Jennifer F" uniqKey="Garcia J" first="Jennifer F" last="Garcia">Jennifer F. Garcia</name><name sortKey="Madhani, Hiten D" sort="Madhani, Hiten D" uniqKey="Madhani H" first="Hiten D" last="Madhani">Hiten D. Madhani</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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