Fujii Tomomi, Inoue Sumiko, Karashima Takahito, Masumoto Michiko, Yamaguchi Hiroko, Kinoshita Sachiko, Muta Koichiro, Hamasaki Naotaka
Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Hospital, Fukuoka 812-8582.
Rinsho Byori. 2003 Sep;51(9):839-46.
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.
对源自恶性细胞的mRNA进行定量分析，有助于评估白血病状态。在本研究中，我们开发了利用实时PCR检测系统（LightCycler）的RT-PCR方法，用于定量慢性髓性白血病患者外周血和骨髓中的bcr/abl嵌合基因。提取的RNA总量以β-肌动蛋白基因作为内标进行校正。每个基因的批内变异系数和批间变异系数在1.7 - 26.0%范围内，这表明其定量比竞争性PCR方法更精确。在使用来自正常受试者的三个样本从采血到bcr基因定量的情况下，检测的变异系数在7.7 - 27.6%范围内。bcr/abl和WT1基因分别可以从10²到10⁸拷贝以及10到10⁵拷贝进行线性测量。使用LightCycler系统进行实时PCR检测时，在2×10⁶个总细胞中有2×10³个K562细胞显示出bcr/abl基因，而使用巢式PCR方法在2×10⁶个总细胞中可以检测到2×10¹个K562细胞。在对七个临床样本的检测中，五个样本显示出bcr/abl和WT1基因，而另外两名骨髓移植患者和一名正常受试者的样本未检测到。这一结果表明我们的定量方法反映了慢性髓性白血病患者的临床分期。
