Differential display protocol with selected primers that preferentially isolates mRNAs of moderate- to low-abundance in a microscopic system.
Identifieur interne : 003E29 ( Main/Exploration ); précédent : 003E28; suivant : 003E30Differential display protocol with selected primers that preferentially isolates mRNAs of moderate- to low-abundance in a microscopic system.
Auteurs : O C Ikonomov [États-Unis] ; M H JacobSource :
- BioTechniques [ 0736-6205 ] ; 1996.
Descripteurs français
- KwdFr :
- MESH :
- isolement et purification : ARN messager, Corps ciliaire.
- métabolisme : Amorces ADN, Corps ciliaire.
- Animaux, Autoradiographie, Banque de gènes, Embryon de poulet, Ganglions, Réaction de polymérisation en chaîne, Séquence nucléotidique, Transcription génétique.
English descriptors
- KwdEn :
- MESH :
- chemical , isolation & purification : RNA, Messenger.
- chemical , metabolism : DNA Primers.
- chemistry : Ganglia.
- innervation : Ciliary Body.
- metabolism : Ciliary Body.
- methods : Polymerase Chain Reaction.
- Animals, Autoradiography, Base Sequence, Chick Embryo, Gene Library, Transcription, Genetic.
Abstract
A modified reverse transcription polymerase chain reaction (RT-PCR)-based differential display procedure with selected primers (SPR) was developed to increase the bias toward isolating moderate- to low-abundance transcripts that are differentially expressed during synapse formation in a microscopic neuronal system, the embryonic chicken ciliary ganglion. Major modifications, in comparison with available arbitrarily primed RT-PCR protocols, include the use of (i) experimentally selected primer pairs (50% GC-rich 15-21-mers) that avoid the amplification of highly abundant ribosomal and mitochondrial transcripts; (ii) a higher PCR annealing temperature (50 degrees C instead of 40 degrees C); (iii) selection of sequencing gel bands that are dependent on the two primers for amplification; (iv) tests for reproducibility by SPR amplification of independent sets of RNA extractions and Southern blot analysis of the products with an isolated radiolabeled clone; and (v) quantitative RT-PCR, instead of Northern blot analysis, to confirm the differential expression of individual cDNAs. Thirty-six cDNAs were isolated and sequenced using SPR. None showed significant homology to highly abundant transcripts. In contrast, when no criterion for primer or band selection was applied, 22% of 55 cDNAs were identical to ribosomal and mitochondrial transcripts. Reproducible amplification of 9 out of 10 SPR-isolated cDNAs was established by Southern blot analysis. Differential expression was then confirmed for 4 selected sequences by quantitative RT-PCR. Thus, SPR is a reproducible and efficient procedure for identifying differentially regulated transcripts of moderate- to low-abundance in microscopic biological systems.
DOI: 10.2144/96206rr01
PubMed: 8780874
Affiliations:
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preferentially isolates mRNAs of moderate- to low-abundance in a microscopic system.</title><author><name sortKey="Ikonomov, O C" sort="Ikonomov, O C" uniqKey="Ikonomov O" first="O C" last="Ikonomov">O C Ikonomov</name><affiliation wicri:level="2"><nlm:affiliation>Worcester Foundation for Biomedical Research, Shrewsbury, MA, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Worcester Foundation for Biomedical Research, Shrewsbury, MA</wicri:regionArea><placeName><region type="state">Massachusetts</region></placeName></affiliation></author><author><name sortKey="Jacob, M H" sort="Jacob, M H" uniqKey="Jacob M" first="M H" last="Jacob">M H Jacob</name></author></analytic><series><title level="j">BioTechniques</title><idno type="ISSN">0736-6205</idno><imprint><date when="1996" type="published">1996</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Autoradiography</term><term>Base Sequence</term><term>Chick Embryo</term><term>Ciliary Body (innervation)</term><term>Ciliary Body (metabolism)</term><term>DNA Primers (metabolism)</term><term>Ganglia (chemistry)</term><term>Gene Library</term><term>Polymerase Chain Reaction (methods)</term><term>RNA, Messenger (isolation & purification)</term><term>Transcription, Genetic</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ARN messager (isolement et purification)</term><term>Amorces ADN (métabolisme)</term><term>Animaux</term><term>Autoradiographie</term><term>Banque de gènes</term><term>Corps ciliaire (innervation)</term><term>Corps ciliaire (métabolisme)</term><term>Embryon de poulet</term><term>Ganglions ()</term><term>Réaction de polymérisation en chaîne ()</term><term>Séquence nucléotidique</term><term>Transcription génétique</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>RNA, Messenger</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>DNA Primers</term></keywords><keywords scheme="MESH" qualifier="chemistry" xml:lang="en"><term>Ganglia</term></keywords><keywords scheme="MESH" qualifier="innervation" xml:lang="en"><term>Ciliary Body</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>ARN messager</term><term>Corps ciliaire</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Ciliary Body</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Polymerase Chain Reaction</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Amorces ADN</term><term>Corps ciliaire</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Autoradiography</term><term>Base Sequence</term><term>Chick Embryo</term><term>Gene Library</term><term>Transcription, Genetic</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Animaux</term><term>Autoradiographie</term><term>Banque de gènes</term><term>Embryon de poulet</term><term>Ganglions</term><term>Réaction de polymérisation en chaîne</term><term>Séquence nucléotidique</term><term>Transcription génétique</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">A modified reverse transcription polymerase chain reaction (RT-PCR)-based differential display procedure with selected primers (SPR) was developed to increase the bias toward isolating moderate- to low-abundance transcripts that are differentially expressed during synapse formation in a microscopic neuronal system, the embryonic chicken ciliary ganglion. Major modifications, in comparison with available arbitrarily primed RT-PCR protocols, include the use of (i) experimentally selected primer pairs (50% GC-rich 15-21-mers) that avoid the amplification of highly abundant ribosomal and mitochondrial transcripts; (ii) a higher PCR annealing temperature (50 degrees C instead of 40 degrees C); (iii) selection of sequencing gel bands that are dependent on the two primers for amplification; (iv) tests for reproducibility by SPR amplification of independent sets of RNA extractions and Southern blot analysis of the products with an isolated radiolabeled clone; and (v) quantitative RT-PCR, instead of Northern blot analysis, to confirm the differential expression of individual cDNAs. Thirty-six cDNAs were isolated and sequenced using SPR. None showed significant homology to highly abundant transcripts. In contrast, when no criterion for primer or band selection was applied, 22% of 55 cDNAs were identical to ribosomal and mitochondrial transcripts. Reproducible amplification of 9 out of 10 SPR-isolated cDNAs was established by Southern blot analysis. Differential expression was then confirmed for 4 selected sequences by quantitative RT-PCR. Thus, SPR is a reproducible and efficient procedure for identifying differentially regulated transcripts of moderate- to low-abundance in microscopic biological systems.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Massachusetts</li></region></list><tree><noCountry><name sortKey="Jacob, M H" sort="Jacob, M H" uniqKey="Jacob M" first="M H" last="Jacob">M H Jacob</name></noCountry><country name="États-Unis"><region name="Massachusetts"><name sortKey="Ikonomov, O C" sort="Ikonomov, O C" uniqKey="Ikonomov O" first="O C" last="Ikonomov">O C Ikonomov</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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