Quantitative analysis of human multidrug resistance 1 (MDR1) gene expression by nonisotopic competitive reverse transcriptase polymerase chain reaction assay.
We have established competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay for the quantification of MDR1 mRNA encoding P-glycoprotein (P-gp) by analyzing leukemia sublines of MOLT-3 with various expression of MDR1. The expression was quantified by simultaneous RT-PCR of cellular RNA with decreasing amounts of heterologous competitor RNA, which shares the MDR1 primer sequences with the cellular MDR1 mRNA, but yields a different-sized PCR product. This allows resolution of the amplified cDNA fragments. The amounts of MDR1 mRNA measured by the assay were accurate and reproducible over wide range, and were determined as 31.6, 100, and 316 amol/microgram total RNA in MOLT-3/TMQ70, MOLT-3/ TMQ800, and MOLT-3/VCR1,000, respectively. The relative ratio of MDR1 mRNA measured by the competitive RT-PCR among three sublines was similar to that of MDR1 transcript determined by Northern analysis (1:4:12) and to that of P-gp measured by flow cytometry (FCM) analysis. In mononuclear cells from patients with leukemia, MDR1 mRNA could be sufficiently quantified by the competitive RT-PCR established, while FCM assay could scarcely detet P-gp. This study demonstrated that the competitive RT-PCR assay using heterologous competitor RNA is a rapid, reliable, and non-radioactive procedure and is acceptable for the evaluation of MDR1 expression in clinical samples.
Kobayashi
H
H
Department of Laboratory Medicine, National Defense Medical College, Saitama, Japan.
Takemura
Y
Y
Miyachi
H
H
Kawabata
M
M
Mori
S
S
Kawai
Y
Y
Furihata
K
K
Sekiguchi
S
S
Watanabe
K
K
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis; D000970:Antineoplastic Agents; D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D019450:Genes, MDR / Q000235:genetics*; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase