en Medical Biology Medical Biology مقاله پژوهشی Original Research Article <span style="font-size:12pt"><span style="font-family:&quot;Times New Roman&quot;,&quot;serif&quot;"><strong>Background</strong>: 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.</span></span><br> <span style="font-size:12pt"><span style="font-family:&quot;Times New Roman&quot;,&quot;serif&quot;"><strong>Methods</strong>: 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 &beta;-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.</span></span><br> <span style="font-size:12pt"><span style="font-family:&quot;Times New Roman&quot;,&quot;serif&quot;"><strong>Results</strong>: 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.</span></span><br> <span style="font-size:12pt"><span style="font-family:&quot;Times New Roman&quot;,&quot;serif&quot;"><strong>Conclusion</strong>: This preliminary assessment shows sufficient specificity and reliability for detecting the PML-RARA bcr1 variant, providing a foundation for further validation in the future.</span></span><br> &nbsp; Key words: Acute Promyelocytic Leukemia (APL), PML-RARA fusion, bcr1 variant, Translocation t (15,17) 9 9 http://journal.zums.ac.ir/browse.php?a_code=A-10-7454-1&slc_lang=en&sid=1 Shahla Rahmani shahlarahmani7075@gmail.com 5200319475328460088374 5200319475328460088374 No Department of Medical Laboratory Sciences, School of Paramedical, Kermanshah University of Medical Sciences, Kermanshah, Iran MohammadHossein Mohammadi DrMohammadi@sbmu.ac.ir 5200319475328460088375 5200319475328460088375 No Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran Bijan Soleimani bijansoleimani@gmail.com 5200319475328460088376 5200319475328460088376 No Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences Kamran Mansouri kamranmansouri@gmail.com 5200319475328460088377 5200319475328460088377 No Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences Ali Maleki maleki.hem@gmail.com 5200319475328460088378 5200319475328460088378 Yes Department of Molecular Medicine, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran