Open AccessQuantitative analysis of human multidrug resistance 1 ( MDR1 ) gene expression by nonisotopic competitive reverse transcriptase polymerase chain reaction assay
Author(s) -
Kobayashi Hiroyuki,
Takemura Yuzuru,
Miyachi Hayato,
Kawabata Masaki,
Mori Shigehisa,
Kawai Yohko,
Furihata Kenichi,
Sekiguchi Susumu,
Watanabe Kiyoaki
Publication year - 1997
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/(sici)1098-2825(1997)11:5<258::aid-jcla4>3.0.co;2-3
Subject(s) - microbiology and biotechnology , reverse transcriptase , primer (cosmetics) , biology , messenger rna , rna , real time polymerase chain reaction , complementary dna , polymerase chain reaction , reverse transcription polymerase chain reaction , heterologous , gene expression , gene , chemistry , biochemistry , organic chemistry
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/μg total RNA in MOLT‐3/TMQ 70 , MOLT‐3/TMQ 800 , and MOLT‐3/VCR 1,000 , 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 detect 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. J. Clin. Lab. Anal. 11:258–266, 1997. © 1997 Wiley‐Liss, Inc. No abstract.