Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Base Sequence
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Blotting, Southern
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DNA Primers
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DNA, Complementary
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Deoxyuracil Nucleotides
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Digoxigenin / analogs & derivatives
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Electrophoresis, Agar Gel
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Humans
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Luminescent Measurements
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RNA, Messenger / analysis
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RNA, Messenger / genetics
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Receptors, Estrogen / genetics
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Reference Standards
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Reverse Transcriptase Polymerase Chain Reaction / methods*
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Sensitivity and Specificity
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Tumor Cells, Cultured
Substances
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DNA Primers
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DNA, Complementary
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Deoxyuracil Nucleotides
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RNA, Messenger
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Receptors, Estrogen
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digoxigenin-11-deoxyuridine triphosphate
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Digoxigenin