[Real-time PCR quantification of bcr/abl chimera and WT1 genes in chronic myeloid leukemia]

Abstract

Quantification of mRNAs deriving from malignant cells is useful for estimating leukemic states. In this study, we have developed RT-PCR methods using real-time PCR detection system, a LightCycler, for quantification of bcr/abl chimerical genes in peripheral blood and bone marrow of chronic myeloid leukemia patients. Total amounts of RNA extracted were corrected using beta-actin gene as an internal standard. The coefficients of variation of intra-assay variation and inter-assay variation for each gene were within a range of 1.7-26.0% which showed more precise quantification than the competitive PCR method. The coefficients of variation of assay are within a range of 7.7-27.6% in the case of using three samples of normal subjects from blood collecting to quantification of bcr gene. Bcr/abl and WT1 genes could be measured from 10(2) to 10(8) copies and 10 to 10(5) copies with linearity, respectively. Using real-time PCR detection with LightCycler system, 2 x 10(3) K562 cells among 2 x 10(6) total cells demonstrated the bcr/abl gene, while 2 x 10(1) K562 cells among 2 x 10(6) total cells could be detected using the nested PCR method. In tests of seven clinical samples, five samples demonstrated bcr/abl and WT1 genes, while those in two other patients after bone marrow transplantation and a normal subject could not detected. This result suggests that our quantitative method reflect the clinical stages of CML patients.