Quantitative polymerase chain reaction analysis of mdr1 mRNA in multiple myeloma cell lines and clinical specimens.
We have designed a new polymerase chain reaction (PCR) protocol for the quantitation of mdr1 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 mdr1 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 mdr1 expression in a multiple myeloma cell line, 8226/S, its drug resistant variants 8226/dox6 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 mdr1 mRNA. 8226/dox6 is 10-fold resistant to doxorubicin, and expresses 32 mdr1 mRNA/10 pg cellular RNA. 8226/dox40 is 140-fold resistant to doxorubicin, and expresses 890 mdr1 mRNA/10 pg cellular RNA. Seven of the 10 patients had levels of mdr1 mRNA expression below that seen in the multidrug-resistant, human multiple myeloma cell line, 8226/dox6. Three patients had levels of mdr1 expression comparable to those seen in 8226/dox6. No patient had levels of mdr1 expression close to that seen in 8226/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 mdr1 expression in cell lines and clinical specimens.
Futscher
B W
BW
Arizona Cancer Center, Bone Marrow Transplant Program, Tucson 85724.
Blake
L L
LL
Gerlach
J H
JH
Grogan
T M
TM
Dalton
W S
WS
D001483:Base Sequence; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance; D004354:Drug Screening Assays, Antitumor; D005813:Genes, Synthetic / Q000235:genetics; D006801:Humans; D015448:Leukemia, B-Cell / Q000188:drug therapy / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000235:genetics; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000188:drug therapy / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D004317:Doxorubicin