A Non-Radioactive Method for Inexpensive Quantitative RT-PCR

We present a novel method for quantitative RT-PCR that involves direct incorporation of digoxigenin-11-dUTP (DIG-dUTP) during amplification of cDNAs, separation of RT-PCR products by agarose gel electrophoresis, Southern transfer to a nylon membrane, and chemiluminescent detection with an anti-DIG a...

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Veröffentlicht in:	Biological chemistry 1999-06, Vol.380 (6), p.695-697
Hauptverfasser:	Maggiolini, M., Donzé, O., Picard, D.
Format:	Artikel
Sprache:	eng
Schlagworte:	Base Sequence Blotting, Southern Deoxyuracil Nucleotides Digoxigenin - analogs & derivatives DNA Primers DNA, Complementary Electrophoresis, Agar Gel Humans Luminescent Measurements Receptors, Estrogen - genetics Reference Standards Reverse Transcriptase Polymerase Chain Reaction - methods RNA, Messenger - analysis RNA, Messenger - genetics Sensitivity and Specificity Tumor Cells, Cultured
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Beschreibung
Zusammenfassung:	We present a novel method for quantitative RT-PCR that involves direct incorporation of digoxigenin-11-dUTP (DIG-dUTP) during amplification of cDNAs, separation of RT-PCR products by agarose gel electrophoresis, Southern transfer to a nylon membrane, and chemiluminescent detection with an anti-DIG antibody. The whole procedure can be done in about a day and has the following advantages: It is highly sensitive, specificity is confirmed by monitoring the size of the RT-PCR product, it is non-radioactive, quantitative, and does not require expensive specialized equipment.
ISSN:	1431-6730 1437-4315
DOI:	10.1515/BC.1999.086