Whole blood and leukocyte RNA isolation for gene expression analyses
Whole blood and leukocyte RNA isolation for gene expression analyses
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DOI：
10.1152/physiolgenomics.00020.2004
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发表时间：
2004-11-17
影响因子：
4.6
通讯作者：
Tompkins, RG
中科院分区：
文献类型：
--
作者：
Feezor, RJ;Baker, HV;Tompkins, RG
临床医学中基因表达数据的分析一直受到缺乏对用于RNA的收集、处理和标记的公认方法的批判性评价的困扰。在本报告中，两种常用的技术分离RNA从全血或其白细胞室的可靠性进行了比较，通过检查其重现性，方差和信噪比。从健康受试者获得全血，并且未处理或用葡萄球菌肠毒素B（SE B）离体刺激。还从创伤患者获得血液样品，但未用SEB离体刺激。使用PAXgene专有血液采集系统从全血中或从分离的白细胞中分离总RNA。将生物素标记的cRNA与Affyssin基因芯片杂交。用这两种技术对健康受试者重复进行基因表达测量的Pearson相关系数非常好，超过0.985。然而，包括分层聚类分析在内的无监督分析显示，RNA分离方法导致的基因表达差异比SEB刺激或不同创伤患者之间的差异更大。来自健康受试者的SEB刺激和未刺激血液之间差异的组内相关性（信噪比的测量）在白细胞来源的样本中显著高于全血：0.75 vs. 0.46（P = 0.002）。在P < 0.001的显著性水平下，用白细胞分离区分SEB刺激的和未刺激的血液的探针组是用PAXgene区分SEB刺激的和未刺激的血液的探针组的两倍。研究结果表明，从全血中分离RNA的方法是使用微阵列分析的临床研究设计中的一个关键变量。
The analysis of gene expression data in clinical medicine has been plagued by the lack of a critical evaluation of accepted methodologies for the collection, processing, and labeling of RNA. In the present report, the reliability of two commonly used techniques to isolate RNA from whole blood or its leukocyte compartment was compared by examining their reproducibility, variance, and signal-to-noise ratios. Whole blood was obtained from healthy subjects and was either untreated or stimulated ex vivo with Staphylococcus enterotoxin B (SEB). Blood samples were also obtained from trauma patients but were not stimulated with SEB ex vivo. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system or from isolated leukocytes. Biotin-labeled cRNA was hybridized to Affymetrix GeneChips. The Pearson correlation coefficient for gene expression measurements in replicates from healthy subjects with both techniques was excellent, exceeding 0.985. Unsupervised analyses, including hierarchical cluster analysis, however, revealed that the RNA isolation method resulted in greater differences in gene expression than stimulation with SEB or among different trauma patients. The intraclass correlation, a measure of signal-to-noise ratio, of the difference between SEB-stimulated and unstimulated blood from healthy subjects was significantly higher in leukocyte-derived samples than in whole blood: 0.75 vs. 0.46 ( P = 0.002). At the P < 0.001 level of significance, twice as many probe sets discriminated between SEB-stimulated and unstimulated blood with leukocyte isolation than with PAXgene. The findings suggest that the method of RNA isolation from whole blood is a critical variable in the design of clinical studies using microarray analyses.