Quantitative assessment of gene expression in highly purified hematopoietic cells using real-time reverse transcriptase polymerase chain reaction
Quantitative assessment of gene expression in highly purified hematopoietic cells using real-time reverse transcriptase polymerase chain reaction
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DOI：
10.1016/s0301-472x(02)00787-7
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发表时间：
2002-05-01
影响因子：
2.6
通讯作者：
Raymakers, RAP
中科院分区：
文献类型：
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作者：
Raaijmakers, MHGP;van Emst, L;Raymakers, RAP
Objective.定量评估干细胞中的基因表达对于理解正常和恶性造血的分子事件是必不可少的。本研究的目的是开发一种精确定量高纯度CD 34（+）CD 38（-）干细胞群中小亚群基因表达的方法。采用实时荧光定量逆转录聚合酶链反应（RT-PCR）技术，对300例CD 34（+）和CD 38（-）细胞中管家基因和耐药基因的表达进行定量分析。在细胞系中的验证实验表明，使用300细胞实时定量RT-PCR进行的基因扩增具有高效性、代表性和可重复性。在稀释实验中和通过检测低拷贝基因PBGD证实了灵敏度。GAPDH是正常和白血病CD 34（+）CD 38（-）细胞中有用的参考基因。相比之下，18 S rRNA含量在这些人群中变化100倍至1000倍。白血病CD 34（+）CD 38（+）细胞18 SrRNA表达明显低于正常CD 34（+）D38（+）细胞（P = 0.002）。与CD 34（+）D38（+）细胞相比，正常CD 34（+）D38（-）细胞中MDR-1（18倍，p <0.0005）、MRP-1（3.8倍，p < 0.05）和LRP（1.8倍，NS）的表达更高。实时定量RT-PCR是精确定量造血细胞小亚群中基因表达的有价值的工具。使用这种方法，我们显示了18 S作为这些祖细胞中的参考基因的不适当性和造血早期耐药相关基因的下调。(C)2002年国际实验血液学学会。出版社：Elsevier Science Inc.
Objective. Quantitative assessment of gene expression in stem cells is essential for understanding the molecular events underlying normal and malignant hematopoiesis. The aim of the present study was to develop a method for precise quantitation of gene expression in small subsets of highly purified CD34(+)CD38(-) stem cell populations.Materials and Methods. Real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to quantitate housekeeping and drug resistance gene expression in cDNA obtained from 300 CD34(+)CD38(-) cells without cDNA amplification or nested PCR techniques.Results. Validation experiments in cell lines showed efficient, representative and reproducible gene amplification using 300-cell real-time quantitative RT-PCR. Sensitivity was confirmed in dilutional experiments and by detection of the low-copy gene PBGD. GAPDH was found to be a useful reference gene in normal and leukemic CD34(+)CD38(-) cells. In contrast, 18S rRNA content varied 100-fold to 1000-fold in these populations. Moreover, expression of 18S rRNA was significantly lower in leukemic CD34(+)CD38(+) cells compared to normal CD34(+)D38(+) cells (P = 0.002). Expression of MDR-1 (18-fold, p < 0.0005), MRP-1 (3.8-fold, p < 0.05), and LRP (1.8-fold, NS) was higher in normal CD34(+)D38(-) compared to CD34(+)D38(+) cells.Conclusions. Real-time quantitative RT-PCR is a valuable tool for precise quantitation of gene expression in small subsets of hematopoietic cells. Using this method, we showed the inappropriateness of 18S as a reference gene in these progenitors and the down-regulation of drug-resistance-related genes early in hematopoiesis. (C) 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.