QUANTITATIVE POLYMERASE CHAIN-REACTION ANALYSIS OF MDR1 MESSENGER-RNA IN MULTIPLE-MYELOMA CELL-LINES AND CLINICAL SPECIMENS

We have designed a new polymerase chain reaction (PCR) protocol for the quantitation of mdr l mRNA in cell lines and clinical specimens. This protocol uses an in vitro-generated RNA molecule as an internal standard. This synthetic RNA contains the same mdr l primer sequences as the cellular mRNA, but yields a different-sized PCR product after amplification. Since a single primer set is used in quantitation, differences in primer efficiency are not a concern. We have used this assay to measure mdr l expression in a multiple myeloma cell line, 8226/5, its drug resistant variants 82261dox6 and 8226/dox40, and tumor samples from 10 patients with B-cell malignancies (9 multiple myeloma, 1 chronic lymphocytic leukemia). 8226/S does not express mdr l mRNA. 8226/dox6 is 10-fold resistant to doxorubicin, and expresses 32 mdr l mRNA/10 pg cellular RNA. 8226/dox4O is 140-fold resistant to doxorubicin, and expresses 890 mdr l mRNA/10 pg cellular RNA. Seven of the 10 patients had levels of mdr l mRNA expression below that seen in the multidrug-resistant, human multiple myeloma cell line, 8226/dox6. Three patients had levels of mdr l expression comparable to those seen in 8226/dox6. No patient had levels of mdr l expression close to that seen in 822 6/dox40. Sample RNA integrity is assured by PCR analysis of a different, ubiquitous, cell cycle independent, histone variant, H3.3. This assay will be useful for studying low level mdr l expression in cell lines and clinical specimens. © 1993 Academic Press, Inc.