Staal F J T, van der Burg M, Wessels L F A, Barendregt B H, Baert M R M, van den Burg C M M, van Huffel C, Langerak A W, van der Velden V H J, Reinders M J T, van Dongen J J M
Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
Leukemia. 2003 Jul;17(7):1324-32. doi: 10.1038/sj.leu.2402974.
Microarrays for gene expression profiling are rapidly becoming important research tools for the identification of novel markers, for example, for novel classification of leukemias and lymphomas. Here, we review the considerations and infrastructure for microarray experiments. These considerations are illustrated via a microarray-based comparison of gene expression profiles of paired diagnosis-relapse samples from patients with precursor-B acute lymphoblastic leukemia (ALL), who relapsed during therapy or after completion of treatment. Initial experiments showed that several seemingly differentially expressed genes were actually derived from contaminating non-leukemic cells, particularly myeloid cells and T-lymphocytes. Therefore, we purified the ALL cells of the diagnosis and relapse samples if their frequency was lower than 95%. Furthermore, we observed in earlier studies that extra RNA amplification leads to skewing of particular gene transcripts. Sufficient (non-amplified) RNA of purified and paired diagnosis-relapse samples was obtained from only seven cases. The gene expression profiles were evaluated with Affymetrix U95A chips containing 12 600 human genes. These diagnosis-relapse comparisons revealed only a small number of genes (n=6) that differed significantly in expression: mostly signaling molecules and transcription factors involved in cell proliferation and cell survival were highly upregulated at relapse, but we did not observe any increase in drug-resistance markers. This finding fits with the observation that tumors with a high proliferation index have a poor prognosis. The genes that changed between diagnosis and relapse are currently not in use as diagnostic or disease progression markers, but represent potential new markers for such applications. Leukemia (2003) 17, 1324-1332. doi:10.1038/sj.leu.2402974
用于基因表达谱分析的微阵列正迅速成为鉴定新型标志物的重要研究工具，例如用于白血病和淋巴瘤的新型分类。在此，我们综述微阵列实验的注意事项和基础设施。通过对前体B急性淋巴细胞白血病（ALL）患者配对的诊断-复发样本的基因表达谱进行基于微阵列的比较来说明这些注意事项，这些患者在治疗期间或治疗完成后复发。初步实验表明，一些看似差异表达的基因实际上来源于污染的非白血病细胞，特别是髓系细胞和T淋巴细胞。因此，如果诊断和复发样本中ALL细胞的频率低于95%，我们就对其进行纯化。此外，我们在早期研究中观察到额外的RNA扩增会导致特定基因转录本的偏差。仅从7例患者中获得了纯化的配对诊断-复发样本的足够（未扩增）RNA。用包含12600个人类基因的Affymetrix U95A芯片评估基因表达谱。这些诊断-复发比较仅揭示了少数在表达上有显著差异的基因（n = 6）：复发时，大多参与细胞增殖和细胞存活的信号分子和转录因子高度上调，但我们未观察到耐药标志物有任何增加。这一发现与高增殖指数的肿瘤预后不良的观察结果相符。诊断和复发之间发生变化的基因目前未用作诊断或疾病进展标志物，但代表了此类应用的潜在新标志物。《白血病》（2003年）17卷，1324 - 1332页。doi:10.1038/sj.leu.2402974
