In situ Localization of the Human Multidrug‐resistance Gene mRNA Using Thymine‐Thymine Dimerized Single‐stranded cDNA

In order to detect the mRNA transcribed from the multidrug‐resistance gene (MDR1), thymine‐thymine (T‐T) dimerized single‐stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used...

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Bibliographic Details
Published in:Cancer science 1990-09, Vol.81 (9), p.949-955
Main Authors: Sugawara, Isamu, Koji, Takehiko, Ueda, Kazumitsu, Pastan, Ira, Gottesman, Michael M., Nakane, Paul K., Mori, Shigeo
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal
ATP Binding Cassette Transporter, Subfamily B, Member 1
Biological and medical sciences
Cytoplasm
Deoxyribonucleic acid
DNA
DNA - genetics
DNA probes
DNA Probes - immunology
DNA, Single-Stranded
Drug Resistance
Fetuses
Gastric cancer
Gene Expression
Humans
Hybridization
Immunoglobulin G
Immunohistochemistry
In situ hybridization
In Vitro Techniques
Localization
MDR1 gene
MDR1 protein
Medical sciences
Membrane Glycoproteins - genetics
mRNA
Nuclease
Nucleic Acid Hybridization
Peroxidase
Pyrimidine Dimers - immunology
RNA, Messenger - metabolism
Sonication
Thymine
Thymine‐thymine dimerized cDNA
Toxicology
Tumor Cells, Cultured
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Summary:In order to detect the mRNA transcribed from the multidrug‐resistance gene (MDR1), thymine‐thymine (T‐T) dimerized single‐stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T‐T dimerized single‐stranded DNA (300–400 bases) so that they could penetrate well into the cytoplasm. The hybridized T‐T DNA was detected immunohisto‐chemically using rabbit anti‐T‐T DNA antibody (Ab) and peroxidase‐labeled goat anti‐rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug‐resistant cell lines K562/ADM, CEM/VLB, 2780ad, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene.
ISSN:0910-5050
1347-9032
1349-7006
1876-4673
DOI:10.1111/j.1349-7006.1990.tb02672.x