A diagnostic tool for monitoring multidrug resistance expression in human tumor tissues

TY - JOUR

T1 - A diagnostic tool for monitoring multidrug resistance expression in human tumor tissues

AU - Schwarzenbach, Heidi

PY - 2002/9/1

Y1 - 2002/9/1

N2 - Studies on multidrug resistance (MDR) require a sensitive and quantitative assay of mRNA expression in clinical tumor samples. Based on the small size, heterogenity, and the possibility of partial degradation of clinical specimens, unambiguous data are often difficult to obtain. The aim of the present study was to develop a multiplex polymerase chain reaction (PCR) in combination with nested PCR for quantitative analyses of mRNA expression of MDR1, MRP (multidrug resistance protein), and DNA topoisomerase IIalpha in small amounts of tumor tissue. RNA samples extracted from the human cell line RPMI 8226 and its MDR sublines 8226/Dox6 and DOXint40c, that overexpress MDR1 and MRP, respectively, were used as model substrates. In the first step, cDNAs of the three genes as well as of the housekeeping gene beta-actin were simultaneously amplified in single tubes using 20 cycles of PCR after random-primed reverse transcription. When necessary, a second amplification step of the preamplified PCR products was employed using nested primer pairs. Primer competition was evaluated by analyses of serially diluted amounts of cDNA and at different numbers of PCR cycles. Based on the results obtained, this multiplex/nested PCR approach may provide a base for quantitative analyses of MDR1, MRP, and topoisomerase IIalpha mRNA expression in clinical tumor biopsies.

AB - Studies on multidrug resistance (MDR) require a sensitive and quantitative assay of mRNA expression in clinical tumor samples. Based on the small size, heterogenity, and the possibility of partial degradation of clinical specimens, unambiguous data are often difficult to obtain. The aim of the present study was to develop a multiplex polymerase chain reaction (PCR) in combination with nested PCR for quantitative analyses of mRNA expression of MDR1, MRP (multidrug resistance protein), and DNA topoisomerase IIalpha in small amounts of tumor tissue. RNA samples extracted from the human cell line RPMI 8226 and its MDR sublines 8226/Dox6 and DOXint40c, that overexpress MDR1 and MRP, respectively, were used as model substrates. In the first step, cDNAs of the three genes as well as of the housekeeping gene beta-actin were simultaneously amplified in single tubes using 20 cycles of PCR after random-primed reverse transcription. When necessary, a second amplification step of the preamplified PCR products was employed using nested primer pairs. Primer competition was evaluated by analyses of serially diluted amounts of cDNA and at different numbers of PCR cycles. Based on the results obtained, this multiplex/nested PCR approach may provide a base for quantitative analyses of MDR1, MRP, and topoisomerase IIalpha mRNA expression in clinical tumor biopsies.

KW - Actins

KW - Blotting, Southern

KW - DNA Primers

KW - DNA Topoisomerases, Type II

KW - DNA, Complementary

KW - Drug Resistance, Multiple

KW - Drug Resistance, Neoplasm

KW - Gene Expression

KW - Humans

KW - Multidrug Resistance-Associated Proteins

KW - Multiple Myeloma

KW - P-Glycoprotein

KW - Polymerase Chain Reaction

KW - RNA, Messenger

KW - Tumor Cells, Cultured

M3 - SCORING: Journal article

C2 - 12234460

VL - 308

SP - 26

EP - 33

JO - ANAL BIOCHEM

JF - ANAL BIOCHEM

SN - 0003-2697

IS - 1

ER -