[Real-time PCR quantification of bcr/abl chimera and WT1 genes in chronic myeloid leukemia].
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.
Fujii
Tomomi
T
Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Hospital, Fukuoka 812-8582.
Inoue
Sumiko
S
Karashima
Takahito
T
Masumoto
Michiko
M
Yamaguchi
Hiroko
H
Kinoshita
Sachiko
S
Muta
Koichiro
K
Hamasaki
Naotaka
N
D002678:Chimera; D016044:Fusion Proteins, bcr-abl / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics*; D012333:RNA, Messenger / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D025721:WT1 Proteins / Q000235:genetics* / Q000302:isolation & purification
D012333:RNA, Messenger; D025721:WT1 Proteins; D016044:Fusion Proteins, bcr-abl