A Quantitative Multistandard Reverse Transcriptase–Polymerase Chain Reaction Assay of the Cystic Fibrosis Transmembrane Conductance Regulator: Its Usefulness in Studying Efficiency of Gene Transfer
Identifieur interne : 000107 ( France/Analysis ); précédent : 000106; suivant : 000108A Quantitative Multistandard Reverse Transcriptase–Polymerase Chain Reaction Assay of the Cystic Fibrosis Transmembrane Conductance Regulator: Its Usefulness in Studying Efficiency of Gene Transfer
Auteurs : Stéphanie Marchand-Pinatel [France] ; Richard Planells [France] ; Marc D. Merten [France] ; Wafa Kammouni [France] ; Catherine Figarella [France]Source :
- Analytical Biochemistry [ 0003-2697 ] ; 1999.
English descriptors
Abstract
Procedures to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels have already been described but are not universally accepted, and many investigators are skeptical about quantification. To be able to accurately monitor gene therapy, we developed a quantitative multistandard RT-PCR method. This was based on the observation that the CFTR and ribosomal phosphoprotein PO (PR-PO) genes have retained important sequence homologies between rat and human species, allowing the use of rat RNA as an internal standard. A mixture of rat and human RNAs is simultaneously reverse-transcribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by restriction analysis. PR-PO is analyzed similarly and serves as a control of template loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) incubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. This method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cystic fibrosis patients.
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DOI: 10.1006/abio.2000.4657
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To be able to accurately monitor gene therapy, we developed a quantitative multistandard RT-PCR method. This was based on the observation that the CFTR and ribosomal phosphoprotein PO (PR-PO) genes have retained important sequence homologies between rat and human species, allowing the use of rat RNA as an internal standard. A mixture of rat and human RNAs is simultaneously reverse-transcribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by restriction analysis. PR-PO is analyzed similarly and serves as a control of template loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) incubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. This method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cystic fibrosis patients.</div></front></TEI><affiliations><list><country><li>France</li></country><region><li>Provence-Alpes-Côte d'Azur</li></region><settlement><li>Marseille</li></settlement></list><tree><country name="France"><region name="Provence-Alpes-Côte d'Azur"><name sortKey="Marchand Pinatel, Stephanie" sort="Marchand Pinatel, Stephanie" uniqKey="Marchand Pinatel S" first="Stéphanie" last="Marchand-Pinatel">Stéphanie Marchand-Pinatel</name></region><name sortKey="Figarella, Catherine" sort="Figarella, Catherine" uniqKey="Figarella C" first="Catherine" last="Figarella">Catherine Figarella</name><name sortKey="Kammouni, Wafa" sort="Kammouni, Wafa" uniqKey="Kammouni W" first="Wafa" last="Kammouni">Wafa Kammouni</name><name sortKey="Merten, Marc D" sort="Merten, Marc D" uniqKey="Merten M" first="Marc D." last="Merten">Marc D. 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