Nonisotopic competitive RT-PCR assay to measure MDR1 gene expression.
We report an original application of competitive reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of MDR1 mRNA in clinical specimens by simultaneous reverse transcription and PCR amplification of cellular RNA with decreasing amounts of an internal standard. The competitor RNA shares the same MDR1 primer sequences as the cellular mRNA, but yields a different-sized PCR product. This allows resolution of the amplified cDNA fragments after agarose gel electrophoresis and ethidium bromide staining. The concentration of MDR1 mRNA is derived from the ratio between the intensities of the bands corresponding to the amplified products. We have used this assay to measure MDR1 expression in breast carcinomas and assessed the precision, sensitivity, and accuracy of the method. Competitive RT-PCR is a simple, highly specific, nonradioactive procedure for the quantification of MDR1 mRNA and is particularly suitable for use in the clinical laboratory.
Debuire
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Service de Biochimie et Biologie Moléculaire, Hôpital Paul Brousse, Villejuif, France.
Sol
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Lemoine
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May
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D001483:Base Sequence; D001667:Binding, Competitive; D001943:Breast Neoplasms; D002461:Cell Line, Transformed; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase