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Quantitative analysis of the expression of apoptosis-related genes
1Department of Clinical Laboratory, Higher Medical Institute, Plovdiv, Bulgaria.
Background:
The development and subsequent introduction of current molecular biology techniques allows the elucidation of some of the mechanisms of the programmed cell death (apoptosis). The overexpression of the genes directly (B-cell leukemia/lymphoma-2, BCL-2) or indirectly (multidrug resistance gene-1, MDR1) involved in the suppression of apoptosis, as well as the low-level expression of their antagonists (BCL-2 associated X-protein, BAX) result in longer survival and the development of drug resistance in malignant cells.
Aim:
The objective of this study was to design our own appropriate primers and to synthesize internal RNA standards and this way to evaluate the rate of expression of the MDR1, BCL-2 and BAX genes using a competitive reverse transcription and polymerase chain reaction (cRT-PCR).
Material And Methods:
Peripheral blood or bone marrow aspirates from 5 patients and 3 healthy controls were used for RNA extraction. Competitive RT-PCR was employed utilizing the constructed specific primers and synthesized internal RNA standards. Following electrophoresis, the PCR products stained with ethidium bromide were evaluated on the "Gel Doc 1000TM" video image system.
Results:
The constructed primers specific for each of the three genes and synthesized internal RNA standards with a suitable deletion allow receiving the yield of well-separated competitive amplicons. The measured band's intensity of light and the calculated standard/patient ratio were employed in the analysis of the MDR1, BCL-2 and BAX gene expression.
Conclusions:
Competitive RT-PCR is a non-radioactive and comparatively rapid molecular biology method suitable for everyday laboratory practice. The possibility for simultaneous evaluation of the expression of several genes could help in the elucidation of some of the mechanisms underlying the prolonged survival of malignant cells.
Insights
This study developed a competitive reverse transcription PCR method to measure gene expression in malignant cells. This technique helps understand how genes like BCL-2, MDR1, and BAX contribute to prolonged survival and drug resistance.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Apoptosis mechanisms are increasingly understood through molecular biology techniques.
- Overexpression of BCL-2 and MDR1, and low BAX expression, promote malignant cell survival and drug resistance.
Purpose of the Study:
- To design primers and synthesize RNA standards for evaluating MDR1, BCL-2, and BAX gene expression.
- To utilize competitive reverse transcription PCR (cRT-PCR) for quantitative gene expression analysis.
Main Methods:
- RNA extraction from patient and healthy control samples (blood/bone marrow).
- Implementation of cRT-PCR with custom primers and internal RNA standards.
- Analysis of PCR products using gel electrophoresis and video imaging systems.
Main Results:
- Successfully generated specific primers and RNA standards for MDR1, BCL-2, and BAX.
- Quantified gene expression by analyzing amplicon band intensity and standard/patient ratios.
Conclusions:
- Competitive RT-PCR is a rapid, non-radioactive method for gene expression analysis.
- Simultaneous gene expression evaluation aids in understanding malignant cell survival mechanisms.