Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR
Real-time PCR is a novel technology recently described to perform quantitative analysis of amplified products. Unlike classical quantitative PCR, this method is easy to standardize, does not required extensive manipulation, and is not reagent intensive, so that the risk of contamination is minimized...
Gespeichert in:
| Veröffentlicht in: | Molecular biotechnology 2000-03, Vol.14 (3), p.189-195 |
|---|---|
| Hauptverfasser: | , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 195 |
|---|---|
| container_issue | 3 |
| container_start_page | 189 |
| container_title | Molecular biotechnology |
| container_volume | 14 |
| creator | KLEIN, D DENIS, M RICORDI, C PASTORI, R. L |
| description | Real-time PCR is a novel technology recently described to perform quantitative analysis of amplified products. Unlike classical quantitative PCR, this method is easy to standardize, does not required extensive manipulation, and is not reagent intensive, so that the risk of contamination is minimized. Therefore, we have chosen reverse transcriptase real-time PCR to quantitate CD95 (Fas) transcripts to test the cleavage efficiency of anti-Fas ribozymes in the mouse insulinoma cell line beta TC-3. Based on the melting-curve analysis of the amplified products, we determined the temperature at which to collect the fluorescent data used for quantification. After constructing a standard curve by plotting the log of the standards' copy number versus their fractional cycle number, the copy numbers of the unknown samples were automatically determined by interpolation of this curve. As we illustrate in this study, it is important, particularly while setting up the technique, to validate the melting-curve profile with standard gel electrophoresis analysis, achieved by matching melting temperature and size of the amplified product. The method is fast and reproducible: Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 hr. The coefficient of variance is 15% in the range of 10(4)-10(6) gene copies. Accordingly, reverse transcriptase (RT) real-time PCR is a technique suitable for screening a large number of ribozymes. |
| doi_str_mv | 10.1385/MB:14:3:189 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_839694130</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>71249399</sourcerecordid><originalsourceid>FETCH-LOGICAL-c404t-a38651ff3a69026f9a2cf0b9acd6bb9643ddddad3809740b87fdef5c4a5992a83</originalsourceid><addsrcrecordid>eNqF0ctrFTEUBvAgiq3VlXsZRHQhoyfv5O7aiy9oUaSuQyZzUlLmcU1mCte_3si9oLjQbE4O_PJB-Ah5SuEN5Ua-vbrYULHhG2rsPXJKpbQtcJD36x00bxUYeUIelXILwKgU_CE5oWAsANhTcn1eCpYy4rQ0c2xy6uYf-xGbMKC_8zfYYIwpJJzCvllLmm6ajHeYCzZL9lMJOe0WX7eMfmiXVF9-2X59TB5EPxR8cpxn5Nv7d9fbj-3l5w-ftueXbRAgltZzoySNkXtlgaloPQsROutDr7rOKsH7enzPDVgtoDM69hhlEF5ay7zhZ-TVIXeX5-8rlsWNqQQcBj_hvBZnuFVWUA5Vvvyn1JQJy639L6RaaQZaV_j8L3g7r3mq33WMK201MFbR6wMKeS4lY3S7nEaf946C-1Weu7pwVDjuanlVPztGrt2I_R_20FYFL47Al-CHWBsIqfx2gmlrFP8JKZ-g_Q</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>236797022</pqid></control><display><type>article</type><title>Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR</title><source>MEDLINE</source><source>SpringerLink Journals</source><creator>KLEIN, D ; DENIS, M ; RICORDI, C ; PASTORI, R. L</creator><creatorcontrib>KLEIN, D ; DENIS, M ; RICORDI, C ; PASTORI, R. L</creatorcontrib><description>Real-time PCR is a novel technology recently described to perform quantitative analysis of amplified products. Unlike classical quantitative PCR, this method is easy to standardize, does not required extensive manipulation, and is not reagent intensive, so that the risk of contamination is minimized. Therefore, we have chosen reverse transcriptase real-time PCR to quantitate CD95 (Fas) transcripts to test the cleavage efficiency of anti-Fas ribozymes in the mouse insulinoma cell line beta TC-3. Based on the melting-curve analysis of the amplified products, we determined the temperature at which to collect the fluorescent data used for quantification. After constructing a standard curve by plotting the log of the standards' copy number versus their fractional cycle number, the copy numbers of the unknown samples were automatically determined by interpolation of this curve. As we illustrate in this study, it is important, particularly while setting up the technique, to validate the melting-curve profile with standard gel electrophoresis analysis, achieved by matching melting temperature and size of the amplified product. The method is fast and reproducible: Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 hr. The coefficient of variance is 15% in the range of 10(4)-10(6) gene copies. Accordingly, reverse transcriptase (RT) real-time PCR is a technique suitable for screening a large number of ribozymes.</description><identifier>ISSN: 1073-6085</identifier><identifier>EISSN: 1559-0305</identifier><identifier>EISSN: 1073-6085</identifier><identifier>DOI: 10.1385/MB:14:3:189</identifier><identifier>PMID: 10890009</identifier><identifier>CODEN: MLBOEO</identifier><language>eng</language><publisher>Totowa, NJ: Humana Press</publisher><subject>Animals ; Biological and medical sciences ; Biotechnology ; Blotting, Northern ; CD95 antigen ; Cytokines - pharmacology ; DNA Primers - chemistry ; DNA Probes - genetics ; DNA, Complementary - genetics ; fas Receptor - genetics ; Fluorescent Dyes ; Fundamental and applied biological sciences. Psychology ; Gene therapy ; Health. Pharmaceutical industry ; Industrial applications and implications. Economical aspects ; insulinoma ; Insulinoma - metabolism ; Melting ; Mice ; Microbiology ; Polymerase chain reaction ; Real time ; Reverse Transcriptase Polymerase Chain Reaction ; Ribozymes ; RNA, Catalytic - metabolism ; RNA, Messenger - metabolism ; Studies ; Temperature ; Tumor Cells, Cultured</subject><ispartof>Molecular biotechnology, 2000-03, Vol.14 (3), p.189-195</ispartof><rights>2000 INIST-CNRS</rights><rights>Humana Press Inc. 2000</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c404t-a38651ff3a69026f9a2cf0b9acd6bb9643ddddad3809740b87fdef5c4a5992a83</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1427986$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10890009$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>KLEIN, D</creatorcontrib><creatorcontrib>DENIS, M</creatorcontrib><creatorcontrib>RICORDI, C</creatorcontrib><creatorcontrib>PASTORI, R. L</creatorcontrib><title>Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR</title><title>Molecular biotechnology</title><addtitle>Mol Biotechnol</addtitle><description>Real-time PCR is a novel technology recently described to perform quantitative analysis of amplified products. Unlike classical quantitative PCR, this method is easy to standardize, does not required extensive manipulation, and is not reagent intensive, so that the risk of contamination is minimized. Therefore, we have chosen reverse transcriptase real-time PCR to quantitate CD95 (Fas) transcripts to test the cleavage efficiency of anti-Fas ribozymes in the mouse insulinoma cell line beta TC-3. Based on the melting-curve analysis of the amplified products, we determined the temperature at which to collect the fluorescent data used for quantification. After constructing a standard curve by plotting the log of the standards' copy number versus their fractional cycle number, the copy numbers of the unknown samples were automatically determined by interpolation of this curve. As we illustrate in this study, it is important, particularly while setting up the technique, to validate the melting-curve profile with standard gel electrophoresis analysis, achieved by matching melting temperature and size of the amplified product. The method is fast and reproducible: Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 hr. The coefficient of variance is 15% in the range of 10(4)-10(6) gene copies. Accordingly, reverse transcriptase (RT) real-time PCR is a technique suitable for screening a large number of ribozymes.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Blotting, Northern</subject><subject>CD95 antigen</subject><subject>Cytokines - pharmacology</subject><subject>DNA Primers - chemistry</subject><subject>DNA Probes - genetics</subject><subject>DNA, Complementary - genetics</subject><subject>fas Receptor - genetics</subject><subject>Fluorescent Dyes</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene therapy</subject><subject>Health. Pharmaceutical industry</subject><subject>Industrial applications and implications. Economical aspects</subject><subject>insulinoma</subject><subject>Insulinoma - metabolism</subject><subject>Melting</subject><subject>Mice</subject><subject>Microbiology</subject><subject>Polymerase chain reaction</subject><subject>Real time</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Ribozymes</subject><subject>RNA, Catalytic - metabolism</subject><subject>RNA, Messenger - metabolism</subject><subject>Studies</subject><subject>Temperature</subject><subject>Tumor Cells, Cultured</subject><issn>1073-6085</issn><issn>1559-0305</issn><issn>1073-6085</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNqF0ctrFTEUBvAgiq3VlXsZRHQhoyfv5O7aiy9oUaSuQyZzUlLmcU1mCte_3si9oLjQbE4O_PJB-Ah5SuEN5Ua-vbrYULHhG2rsPXJKpbQtcJD36x00bxUYeUIelXILwKgU_CE5oWAsANhTcn1eCpYy4rQ0c2xy6uYf-xGbMKC_8zfYYIwpJJzCvllLmm6ajHeYCzZL9lMJOe0WX7eMfmiXVF9-2X59TB5EPxR8cpxn5Nv7d9fbj-3l5w-ftueXbRAgltZzoySNkXtlgaloPQsROutDr7rOKsH7enzPDVgtoDM69hhlEF5ay7zhZ-TVIXeX5-8rlsWNqQQcBj_hvBZnuFVWUA5Vvvyn1JQJy639L6RaaQZaV_j8L3g7r3mq33WMK201MFbR6wMKeS4lY3S7nEaf946C-1Weu7pwVDjuanlVPztGrt2I_R_20FYFL47Al-CHWBsIqfx2gmlrFP8JKZ-g_Q</recordid><startdate>20000301</startdate><enddate>20000301</enddate><creator>KLEIN, D</creator><creator>DENIS, M</creator><creator>RICORDI, C</creator><creator>PASTORI, R. L</creator><general>Humana Press</general><general>Springer Nature B.V</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QL</scope><scope>7QO</scope><scope>7T7</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>Q9U</scope><scope>7X8</scope><scope>7TM</scope></search><sort><creationdate>20000301</creationdate><title>Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR</title><author>KLEIN, D ; DENIS, M ; RICORDI, C ; PASTORI, R. L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c404t-a38651ff3a69026f9a2cf0b9acd6bb9643ddddad3809740b87fdef5c4a5992a83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Blotting, Northern</topic><topic>CD95 antigen</topic><topic>Cytokines - pharmacology</topic><topic>DNA Primers - chemistry</topic><topic>DNA Probes - genetics</topic><topic>DNA, Complementary - genetics</topic><topic>fas Receptor - genetics</topic><topic>Fluorescent Dyes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene therapy</topic><topic>Health. Pharmaceutical industry</topic><topic>Industrial applications and implications. Economical aspects</topic><topic>insulinoma</topic><topic>Insulinoma - metabolism</topic><topic>Melting</topic><topic>Mice</topic><topic>Microbiology</topic><topic>Polymerase chain reaction</topic><topic>Real time</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Ribozymes</topic><topic>RNA, Catalytic - metabolism</topic><topic>RNA, Messenger - metabolism</topic><topic>Studies</topic><topic>Temperature</topic><topic>Tumor Cells, Cultured</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>KLEIN, D</creatorcontrib><creatorcontrib>DENIS, M</creatorcontrib><creatorcontrib>RICORDI, C</creatorcontrib><creatorcontrib>PASTORI, R. L</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Engineering Collection</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>Nucleic Acids Abstracts</collection><jtitle>Molecular biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>KLEIN, D</au><au>DENIS, M</au><au>RICORDI, C</au><au>PASTORI, R. L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR</atitle><jtitle>Molecular biotechnology</jtitle><addtitle>Mol Biotechnol</addtitle><date>2000-03-01</date><risdate>2000</risdate><volume>14</volume><issue>3</issue><spage>189</spage><epage>195</epage><pages>189-195</pages><issn>1073-6085</issn><eissn>1559-0305</eissn><eissn>1073-6085</eissn><coden>MLBOEO</coden><abstract>Real-time PCR is a novel technology recently described to perform quantitative analysis of amplified products. Unlike classical quantitative PCR, this method is easy to standardize, does not required extensive manipulation, and is not reagent intensive, so that the risk of contamination is minimized. Therefore, we have chosen reverse transcriptase real-time PCR to quantitate CD95 (Fas) transcripts to test the cleavage efficiency of anti-Fas ribozymes in the mouse insulinoma cell line beta TC-3. Based on the melting-curve analysis of the amplified products, we determined the temperature at which to collect the fluorescent data used for quantification. After constructing a standard curve by plotting the log of the standards' copy number versus their fractional cycle number, the copy numbers of the unknown samples were automatically determined by interpolation of this curve. As we illustrate in this study, it is important, particularly while setting up the technique, to validate the melting-curve profile with standard gel electrophoresis analysis, achieved by matching melting temperature and size of the amplified product. The method is fast and reproducible: Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 hr. The coefficient of variance is 15% in the range of 10(4)-10(6) gene copies. Accordingly, reverse transcriptase (RT) real-time PCR is a technique suitable for screening a large number of ribozymes.</abstract><cop>Totowa, NJ</cop><pub>Humana Press</pub><pmid>10890009</pmid><doi>10.1385/MB:14:3:189</doi><tpages>7</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1073-6085 |
| ispartof | Molecular biotechnology, 2000-03, Vol.14 (3), p.189-195 |
| issn | 1073-6085 1559-0305 1073-6085 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_839694130 |
| source | MEDLINE; SpringerLink Journals |
| subjects | Animals Biological and medical sciences Biotechnology Blotting, Northern CD95 antigen Cytokines - pharmacology DNA Primers - chemistry DNA Probes - genetics DNA, Complementary - genetics fas Receptor - genetics Fluorescent Dyes Fundamental and applied biological sciences. Psychology Gene therapy Health. Pharmaceutical industry Industrial applications and implications. Economical aspects insulinoma Insulinoma - metabolism Melting Mice Microbiology Polymerase chain reaction Real time Reverse Transcriptase Polymerase Chain Reaction Ribozymes RNA, Catalytic - metabolism RNA, Messenger - metabolism Studies Temperature Tumor Cells, Cultured |
| title | Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-01T12%3A33%3A25IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Assessment%20of%20ribozyme%20cleavage%20efficiency%20using%20reverse%20transcriptase%20real-time%20PCR&rft.jtitle=Molecular%20biotechnology&rft.au=KLEIN,%20D&rft.date=2000-03-01&rft.volume=14&rft.issue=3&rft.spage=189&rft.epage=195&rft.pages=189-195&rft.issn=1073-6085&rft.eissn=1559-0305&rft.coden=MLBOEO&rft_id=info:doi/10.1385/MB:14:3:189&rft_dat=%3Cproquest_cross%3E71249399%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=236797022&rft_id=info:pmid/10890009&rfr_iscdi=true |