یکشنبه 24 خرداد 1405
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Background: Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia characterized by the t(15;17) translocation, generating the PML-RARA fusion gene. This fusion occurs in three primary variants: bcr1, bcr2, and bcr3. In Iran, bcr1 is the most prevalent (~73%), bcr3 accounts for ~27%, and bcr2 is virtually undetected. Given this distribution, developing a locally optimized method for detecting bcr1 is essential for accurate and rapid identification of this fusion and crucial for monitoring patient response.
Methods: A synthetic 130-bp PML-RARA bcr1 fragment was cloned into pUC57, and insertion was analyzed by digestion with XbaI and HindIII and agarose gel electrophoresis. RNA from APL patient samples was reverse-transcribed into cDNA, serving as a template for reverse-transcription quantitative polymerase chain reaction (RT-qPCR) with PML-RARA-specific primers and β-actin as the reference. Each run included APL samples, plasmid-based positive controls, healthy negative controls, and no-template controls. Amplification and melt-curve analysis confirmed assay specificity and reproducibility.
Results: The recombinant pUC57-PML-RARA plasmid was verified by XbaI/HindIII digestion. RT-qPCR showed specific amplification of PML-RARA in plasmid controls (Ct 15-20) and APL patient samples (Ct 22-26), with no amplification in negative controls. Melt curve analysis showed single, sharp peaks, confirming specificity, and agarose gel electrophoresis verified the expected product sizes. Relative quantification indicated approximately 26-fold higher expression in plasmid controls compared to patient samples, with high reproducibility at a 1:20 dilution.
Conclusion: This preliminary assessment shows sufficient specificity and reliability for detecting the PML-RARA bcr1 variant, providing a foundation for further validation in the future.
Methods: A synthetic 130-bp PML-RARA bcr1 fragment was cloned into pUC57, and insertion was analyzed by digestion with XbaI and HindIII and agarose gel electrophoresis. RNA from APL patient samples was reverse-transcribed into cDNA, serving as a template for reverse-transcription quantitative polymerase chain reaction (RT-qPCR) with PML-RARA-specific primers and β-actin as the reference. Each run included APL samples, plasmid-based positive controls, healthy negative controls, and no-template controls. Amplification and melt-curve analysis confirmed assay specificity and reproducibility.
Results: The recombinant pUC57-PML-RARA plasmid was verified by XbaI/HindIII digestion. RT-qPCR showed specific amplification of PML-RARA in plasmid controls (Ct 15-20) and APL patient samples (Ct 22-26), with no amplification in negative controls. Melt curve analysis showed single, sharp peaks, confirming specificity, and agarose gel electrophoresis verified the expected product sizes. Relative quantification indicated approximately 26-fold higher expression in plasmid controls compared to patient samples, with high reproducibility at a 1:20 dilution.
Conclusion: This preliminary assessment shows sufficient specificity and reliability for detecting the PML-RARA bcr1 variant, providing a foundation for further validation in the future.
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