Quantitative Polymerase Chain-Reaction Analysis of mdr1 mRNA in Multiple Myeloma Cell Lines and Clinical Specimens

Bernard W Futscher, L. L. Blake, J. H. Gerlach, T. M. Grogan, W. S. Dalton

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)414-421
Number of pages8
JournalAnalytical Biochemistry
Volume213
Issue number2
DOIs
StatePublished - Sep 1993

Fingerprint

Polymerase chain reaction
Multiple Myeloma
Cells
Cell Line
Polymerase Chain Reaction
Messenger RNA
RNA
Doxorubicin
Assays
B-Cell Chronic Lymphocytic Leukemia
Reaction products
Histones
Amplification
Tumors
Neoplasms
Cell Cycle
B-Lymphocytes
Molecules
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Quantitative Polymerase Chain-Reaction Analysis of mdr1 mRNA in Multiple Myeloma Cell Lines and Clinical Specimens. / Futscher, Bernard W; Blake, L. L.; Gerlach, J. H.; Grogan, T. M.; Dalton, W. S.

In: Analytical Biochemistry, Vol. 213, No. 2, 09.1993, p. 414-421.

Research output: Contribution to journalArticle

Futscher, Bernard W ; Blake, L. L. ; Gerlach, J. H. ; Grogan, T. M. ; Dalton, W. S. / Quantitative Polymerase Chain-Reaction Analysis of mdr1 mRNA in Multiple Myeloma Cell Lines and Clinical Specimens. In: Analytical Biochemistry. 1993 ; Vol. 213, No. 2. pp. 414-421.
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