An In-house Method for Molecular Monitoring of BCR-ABL

Abstract

OBJECTIVE: At present in Turkey, centers that are able to give reliable RQ-PCR BCR-ABL results are limited in number. We aimed to describe a cost-effective, in-house method for BCR-ABL quantification and to illustrate an example for RQ-PCR validation tests. METHODS: BCR-ABL and ABL target sequences were cloned into pJET1.2 vectors; from which calibrators were prepared and used as templates in RQ- PCR reactions to generate standard curves. Dilutions of K562 cells (representing an in vitro simulation of BCR-ABL transcript reduction) were analyzed. RESULTS: Previously determined standard curves were used to calculate the BCR-ABL and ABL copy numbers. Linear BCR-ABL results were obtained. Repetitive experiments showed that our methodology is able to detect one BCR-ABL positive cell in a total of 1x105 cells. CONCLUSION: The approach described in this manuscript is suitable for implementation into any RQ-PCR instrument and/or kit aiming to quantify BCR-ABL transcripts.

Keywords

• chronic myeloid leukemia
• bcr-abl
• real-time pcr
• molecular response