Quantitative analysis of the expression of apoptosis-related genes.

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.