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Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription : a ‘zero-step’ RT-qPCR protocol

Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription : a ‘zero-step’ RT-qPCR protocol

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CHOVANCOVA, Petra, Verena MERK, Andreas MARX, Marcel LEIST, Ramon KRANASTER, 2017. Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription : a ‘zero-step’ RT-qPCR protocol. In: Biology Methods and Protocols. 2(1), bpx008. eISSN 2396-8923

@article{Chovancova2017Rever-39336, title={Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription : a ‘zero-step’ RT-qPCR protocol}, year={2017}, doi={10.1093/biomethods/bpx008}, number={1}, volume={2}, journal={Biology Methods and Protocols}, author={Chovancova, Petra and Merk, Verena and Marx, Andreas and Leist, Marcel and Kranaster, Ramon}, note={Article Number: bpx008} }

<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/39336"> <dc:creator>Leist, Marcel</dc:creator> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2017-06-21T09:55:01Z</dcterms:available> <dcterms:title>Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription : a ‘zero-step’ RT-qPCR protocol</dcterms:title> <dcterms:rights rdf:resource="http://nbn-resolving.de/urn:nbn:de:bsz:352-20150914100631302-4485392-8"/> <dc:language>eng</dc:language> <dc:contributor>Merk, Verena</dc:contributor> <dcterms:issued>2017</dcterms:issued> <dc:creator>Marx, Andreas</dc:creator> <dc:contributor>Leist, Marcel</dc:contributor> <dc:contributor>Kranaster, Ramon</dc:contributor> <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/39336"/> <dc:creator>Kranaster, Ramon</dc:creator> <dc:contributor>Marx, Andreas</dc:contributor> <dc:contributor>Chovancova, Petra</dc:contributor> <dc:creator>Chovancova, Petra</dc:creator> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2017-06-21T09:55:01Z</dc:date> <dcterms:abstract xml:lang="eng">We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and without an isothermal reverse-transcription step. Human neurons (Lund human mesencephalic cells) were lysed at different stages of differentiation, and the lysates were used directly as template for the combined RT-qPCR reaction. We detected a downregulation of a proliferation marker and an up-regulation of neuronal dopaminergic genes expression. We were able to detect the reference gene target from as few as a single cell, demonstrating the application of the method for efficient amplification from small cell numbers. The data were fully in line with those obtained by the standard two-step RT-qPCR from the extracted total RNA. Our ‘zero-step’ RT-qPCR method proved to be simple and reliable with a total time from cell lysis to the end of the qPCR as short as 1.5 h. It is therefore particularly suitable for RT-qPCRs where large numbers of samples must be handled, or where data are required within short time.</dcterms:abstract> <dc:creator>Merk, Verena</dc:creator> </rdf:Description> </rdf:RDF>

Dateiabrufe seit 21.06.2017 (Informationen über die Zugriffsstatistik)

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