Multiplex In-cell Reverse Transcription-Polymerase Chain Reaction for the Simultaneous Detection of p210 and p190 BCR-ABL mRNAs in Chronic Myeloid Leukemia and Philadelphia-Positive Acute Lymphoblastic Leukemia Cell Lines
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Abstract
We designed a novel multiplex in-cell reverse transcription- polymerase chain reaction method for the simultaneous detection and differentiation of p190 and p210 BCR-ABL mRNAs within single cells from the human chronic myeloid leukemia and Philadelphia positive acute lymphoblastic leukemia. Human K562 chronic myeloid leukemia and SUP B-15 Ph+ acute lymphoblastic leukemia cell lines were used as positive controls for p210 and p190 BCR-ABL mRNAs, respectively. HL60 cell line was used as a negative control. After the leukemia cells were fixed and permeabilized, without extracting nucleic acids, the mRNAs were reverse transcribed to cDNAs, and the cDNAs were amplified by multiplex polymerase chain reaction with fluorescent primers specific for p190 and p210 BCR-ABL mRNAs. After transfer onto glass slides by cytospin, the amplified cells were detected by fluorescence microscopy. Fluorescence microscopy after propidium iodide or 4′,6-diamidino-2-phenylindone counterstaining showed that the positive K562 cells exhibited a yellow-green fluorescent cytoplasm around a red nucleus, and that the positive SUP B-15 cells exhibited an orange cytoplasm around a blue nucleus. Only the red or blue nucleus was visible in respective negative HL60 cells. The specificity of amplification was confirmed by the absence of a signal when control experiments were performed either with RNase digestion of mRNA or without reverse transcriptase/Taq polymerase. We conclude that the multiplex in-cell reverse transcription-polymerase chain reaction method is capable of simultaneously detecting and differentiating the p210 and p190 BCR-ABL mRNAs of chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia cells, and that it may be useful in quantitatively monitoring the minimal residual disease during therapy.
Copyright © 2000 by Walter de Gruyter GmbH & Co. KG
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- A Functional Genomic Study of the Effects of Antipsychotic Agent Chlorpromazine in PC12 Cells
- Influence of Glutathione S-Transferase M1 and T1 Genotypes on Larynx Cancer Risk among Korean Smokers
- CYP2D6 Genotyping in Patients on Psychoactive Drug Therapy
- Genotyping of CYP2D6 in Parkinsons's Disease
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- Multiplex In-cell Reverse Transcription-Polymerase Chain Reaction for the Simultaneous Detection of p210 and p190 BCR-ABL mRNAs in Chronic Myeloid Leukemia and Philadelphia-Positive Acute Lymphoblastic Leukemia Cell Lines
Articles in the same Issue
- The Basis of the Medicine of Tomorrow "Validating and Using Pharmacogenomics" Joint IFCC-Roche Diagnostics Conference, Kyoto, Japan, 1619 April 2000
- Diagnostics and the Future of Medicine
- Operomics: Molecular Analysis of Tissues from DNA to RNA to Protein
- Idiosyncratic Reactions to Drugs: Can Medicine Response Profiles Provide a Dynamic Drug Surveillance System?
- Hunting for Disease Genes in Multi-Functional Diseases
- Familial Studies on the Genetics of Cardiovascular Diseases: the Stanislas Cohort
- Quantitative PCR
- Gene Amplification as Means for Determining Therapeutic Strategies in Human Cancers
- Apolipoprotein E Polymorphisms and Concentration in Chronic Diseases and Drug Responses
- Angiotensin I-Converting Enzyme Gene Polymorphism and Drug Response
- Drug-Metabolizing Enzymes, Polymorphisms and Interindividual Response to Environmental Toxicants
- Database Analysis and Gene Discovery in Pharmacogenetics
- How to Manage Individualized Drug Therapy: Application of Pharmacogenetic Knowledge of Drug Metabolism and Transport
- P-Glycoprotein and Bioavailability-Implication of Polymorphism
- Cancer Therapy and Polymorphisms of Cytochromes P450
- Polymorphisms in UDP Glucuronosyltransferase Genes: Functional Consequences and Clinical Relevance
- The Human Multidrug Resistance-Associated Protein (MRP) Gene Family: From Biological Function to Drug Molecular Design
- Ethnic Differences in Drug Metabolism
- Hypervariable Region 1 of Hepatitis C Virus Genome and Response to Interferon Therapy
- A Functional Genomic Study of the Effects of Antipsychotic Agent Chlorpromazine in PC12 Cells
- Influence of Glutathione S-Transferase M1 and T1 Genotypes on Larynx Cancer Risk among Korean Smokers
- CYP2D6 Genotyping in Patients on Psychoactive Drug Therapy
- Genotyping of CYP2D6 in Parkinsons's Disease
- Rapid Analysis of CGG Repeat Length in the FMR1 Gene
- Multiplex In-cell Reverse Transcription-Polymerase Chain Reaction for the Simultaneous Detection of p210 and p190 BCR-ABL mRNAs in Chronic Myeloid Leukemia and Philadelphia-Positive Acute Lymphoblastic Leukemia Cell Lines
