Nonradioactive quantification of mdr1 mRNA by polymerase chain reaction amplification coupled with HPLC.
We describe a new strategy for quantifying mRNA by using polymerase chain reaction (PCR) coupled with HPLC. PCR-amplified products are directly analyzed with a specific HPLC column and are quantified by standardization against a housekeeping gene, beta-actin. To evaluate the experimental conditions, we examined the multidrug-resistance-associated mdr1 gene expression in two drug-sensitive cell lines (RPMI-8226 and KB-3-1) and in their drug-resistant sublines. This revealed a significant overexpression of the mdr1 gene in chemoresistant sublines that paralleled the degree of in vitro drug resistance and the amounts of mRNA determined by other methods. Results were consistently reproducible, with imprecision (CV) < 25%. The reverse transcription option PCR/HPLC protocol described here is a sensitive, nonradioactive method for quantifying mdr1 mRNA in cell lines or clinical tumor samples, and can be applied to the simultaneous quantification of several mRNA species.
van Hille
B
B
Department of Research ZLF-301, Kantonsspital Basel, Switzerland.
Lohri
A
A
Reuter
J
J
Herrmann
R
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000199:Actins / Q000235:genetics; D001483:Base Sequence; D002294:Carcinoma, Squamous Cell; D002851:Chromatography, High Pressure Liquid / Q000706:statistics & numerical data; D006801:Humans; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D016133:Polymerase Chain Reaction / Q000706:statistics & numerical data; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase