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 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.
Maggiolini
M
M
Département de Biologie Cellulaire, Université de Genève, Switzerland.
Donzé
O
O
Picard
D
D
D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D018076:DNA, Complementary; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin / Q000031:analogs & derivatives; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008163:Luminescent Measurements; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D003856:Deoxyuracil Nucleotides; D012333:RNA, Messenger; D011960:Receptors, Estrogen; C070202:digoxigenin-11-deoxyuridine triphosphate; D004076:Digoxigenin