Evaluating the feasibility of nucleated cells isolated by lysis method for real-time quantitative RT-PCR / 中国实验血液学杂志

ABSTRACT

This study was aimed to investigate whether difference exists between real time RT-PCR results of nucleated cells isolated by lysis method and mononuclear cells isolated by gradient concentration method. 14 bone marrow samples from leukemia patients (7 samples of AML-M(2), 1 of AML-M(4), 1 of AML-M(4)EO, 1 of AML-M(6), 1 of APL and 3 of CML) were collected. Each sample was divided into 2 parts, and was used to isolate mononuclear cells by Ficoll-Hypaque gradient centrifugation, and other was used to isolate nuclear cells by lysis method. The RNA extraction and detection of internal reference gene ABL for all of samples were performed by RT-PCR, and mRNA expression levels in 3 samples of BCR/ABL, 6 of AML/ETO, 1 of CBFβ-MHY11, 1 of WT1 and 6 PRAME were detected by RT-PCR. The results showed that ABL copies of all samples were over 3 × 10(4), and there was significant difference in ABL copies between each pair of samples by lysis and gradient centrifugation method (p > 0.05). There was also no significant difference in every detected mRNA levels between 14 pair samples (p > 0.05). It is concluded that the lysis method may be useful one for extracting RNA from nuclear cells, and can adopt as a routing detection method for simultaneously extracting RNA from many samples to detect leukemia specific mRNA by using RT-PCR.