Quantitative monitoring of the mRNA expression pattern of the TGF-β-isoforms (β1, β2, β3) during transdifferentiation of hepatic stellate cells using a newly developed real-time SYBR Green PCR
Current methods to determine the mRNA of the TGF-β-isoforms, β1, β2, and β3, are not sensitive enough to detect small alterations in the expression levels. Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-spec...
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| Veröffentlicht in: | Biochemical and biophysical research communications 2002-07, Vol.295 (2), p.330-335 |
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| creator | Wickert, L Steinkrüger, S Abiaka, M Bolkenius, U Purps, O Schnabel, C Gressner, A.M |
| description | Current methods to determine the mRNA of the TGF-β-isoforms, β1, β2, and β3, are not sensitive enough to detect small alterations in the expression levels. Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-specific quantification is the possibility to be abstain from the need to compare results with a house-keeping gene having a different sequence and PCR efficiency. Reproducibility of the results and analytical variances of the real-time PCR assays were tested. In transdifferentiating rat hepatic stellate cells (HSC) the TGF-β1-mRNA was found to be the predominant isoform expressed followed by TGF-β3 and low amounts of TGF-β2-mRNA. An alteration of the TGF-β1,-β2, and -β3 ratio during HSC transdifferentiation could not be detected. Furthermore, the GAPDH mRNA expression varied during HSC activation, and thus is not recommended as a standard in real-time PCR quantifications. |
| doi_str_mv | 10.1016/S0006-291X(02)00669-1 |
| format | Article |
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Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-specific quantification is the possibility to be abstain from the need to compare results with a house-keeping gene having a different sequence and PCR efficiency. Reproducibility of the results and analytical variances of the real-time PCR assays were tested. In transdifferentiating rat hepatic stellate cells (HSC) the TGF-β1-mRNA was found to be the predominant isoform expressed followed by TGF-β3 and low amounts of TGF-β2-mRNA. An alteration of the TGF-β1,-β2, and -β3 ratio during HSC transdifferentiation could not be detected. 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Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-specific quantification is the possibility to be abstain from the need to compare results with a house-keeping gene having a different sequence and PCR efficiency. Reproducibility of the results and analytical variances of the real-time PCR assays were tested. In transdifferentiating rat hepatic stellate cells (HSC) the TGF-β1-mRNA was found to be the predominant isoform expressed followed by TGF-β3 and low amounts of TGF-β2-mRNA. An alteration of the TGF-β1,-β2, and -β3 ratio during HSC transdifferentiation could not be detected. Furthermore, the GAPDH mRNA expression varied during HSC activation, and thus is not recommended as a standard in real-time PCR quantifications.</description><subject>Animals</subject><subject>Base Sequence</subject><subject>Cell Differentiation</subject><subject>DNA Primers</subject><subject>Glyceraldehyde-3-Phosphate Dehydrogenases - genetics</subject><subject>Hepatic stellate cells</subject><subject>Hepatocytes - cytology</subject><subject>Hepatocytes - metabolism</subject><subject>Male</subject><subject>mRNA-quantification</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Protein Isoforms - genetics</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Real-time PCR</subject><subject>Reference Standards</subject><subject>RNA, Messenger - genetics</subject><subject>TGF-β-isoforms</subject><subject>Transforming Growth Factor beta - genetics</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFUctuEzEUtRCIhsIngLxCrcSA7WQeXqEStQGp4pEWCVaWY19Toxl7sD0p_a18A-t8E54kwJKNr3Xvedj3IPSUkpeU0OrVFSGkKhinX04IO833ihf0HppQwknBKJndR5O_kCP0KMbvhFA6q_hDdEQZLQkv2QT9-jRIl2ySya4Bd97Z5IN137A3ON3kzvL9GYaffYAYrXe4lylBcH_G14uLYrspbPTGhy7ik-2GvsDbDRuP6SnWw04sBemitsZAgOyWzfxO4gaynlU4JmhbmQCrXCMe4kiS2MFte4c1rKH1PWgcQLZFsh3gq69vlngRABz-OF8-Rg-MbCM8OdRj9Pni_Hr-trj8sHg3P7ss1LSiqTBNs6o0aWqzog2dUa0YcM7NivOG1UTrqaxLIGSqawIlVayRqirzxqRupJFqeoye73X74H8MEJPobByfLB34IYqa8rrifJaB5R6ogo8xgBF9sJ0Md4ISMcYndvGJMRtBmNjFJ2jmPTsYDKsO9D_WIa8MeL0HQP7m2kIQUVlwCrQNoJLQ3v7H4jc5e6-_</recordid><startdate>20020712</startdate><enddate>20020712</enddate><creator>Wickert, L</creator><creator>Steinkrüger, S</creator><creator>Abiaka, M</creator><creator>Bolkenius, U</creator><creator>Purps, O</creator><creator>Schnabel, C</creator><creator>Gressner, A.M</creator><general>Elsevier Inc</general><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>7X8</scope></search><sort><creationdate>20020712</creationdate><title>Quantitative monitoring of the mRNA expression pattern of the TGF-β-isoforms (β1, β2, β3) during transdifferentiation of hepatic stellate cells using a newly developed real-time SYBR Green PCR</title><author>Wickert, L ; 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Therefore, we established a SYBR Green I-based real-time quantitative PCR procedure with fragment-specific standards. The advantage of gene-specific quantification is the possibility to be abstain from the need to compare results with a house-keeping gene having a different sequence and PCR efficiency. Reproducibility of the results and analytical variances of the real-time PCR assays were tested. In transdifferentiating rat hepatic stellate cells (HSC) the TGF-β1-mRNA was found to be the predominant isoform expressed followed by TGF-β3 and low amounts of TGF-β2-mRNA. An alteration of the TGF-β1,-β2, and -β3 ratio during HSC transdifferentiation could not be detected. Furthermore, the GAPDH mRNA expression varied during HSC activation, and thus is not recommended as a standard in real-time PCR quantifications.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>12150952</pmid><doi>10.1016/S0006-291X(02)00669-1</doi><tpages>6</tpages></addata></record> |
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| subjects | Animals Base Sequence Cell Differentiation DNA Primers Glyceraldehyde-3-Phosphate Dehydrogenases - genetics Hepatic stellate cells Hepatocytes - cytology Hepatocytes - metabolism Male mRNA-quantification Polymerase Chain Reaction - methods Protein Isoforms - genetics Rats Rats, Sprague-Dawley Real-time PCR Reference Standards RNA, Messenger - genetics TGF-β-isoforms Transforming Growth Factor beta - genetics |
| title | Quantitative monitoring of the mRNA expression pattern of the TGF-β-isoforms (β1, β2, β3) during transdifferentiation of hepatic stellate cells using a newly developed real-time SYBR Green PCR |
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