Gnatenko Dmitri V, Dunn John J, McCorkle Sean R, Weissmann David, Perrotta Peter L, Bahou Wadie F
Department of Medicine, Program in Genetics, State University of New York, Stony Brook 11794-8151, USA.
Blood. 2003 Mar 15;101(6):2285-93. doi: 10.1182/blood-2002-09-2797. Epub 2002 Nov 14.
Human platelets are anucleate blood cells that retain cytoplasmic mRNA and maintain functionally intact protein translational capabilities. We have adapted complementary techniques of microarray and serial analysis of gene expression (SAGE) for genetic profiling of highly purified human blood platelets. Microarray analysis using the Affymetrix HG-U95Av2 approximately 12 600-probe set maximally identified the expression of 2147 (range, 13%-17%) platelet-expressed transcripts, with approximately 22% collectively involved in metabolism and receptor/signaling, and an overrepresentation of genes with unassigned function (32%). In contrast, a modified SAGE protocol using the Type IIS restriction enzyme MmeI (generating 21-base pair [bp] or 22-bp tags) demonstrated that 89% of tags represented mitochondrial (mt) transcripts (enriched in 16S and 12S ribosomal RNAs), presumably related to persistent mt-transcription in the absence of nuclear-derived transcripts. The frequency of non-mt SAGE tags paralleled average difference values (relative expression) for the most "abundant" transcripts as determined by microarray analysis, establishing the concordance of both techniques for platelet profiling. Quantitative reverse transcription-polymerase chain reaction (PCR) confirmed the highest frequency of mt-derived transcripts, along with the mRNAs for neurogranin (NGN, a protein kinase C substrate) and the complement lysis inhibitor clusterin among the top 5 most abundant transcripts. For confirmatory characterization, immunoblots and flow cytometric analyses were performed, establishing abundant cell-surface expression of clusterin and intracellular expression of NGN. These observations demonstrate a strong correlation between high transcript abundance and protein expression, and they establish the validity of transcript analysis as a tool for identifying novel platelet proteins that may regulate normal and pathologic platelet (and/or megakaryocyte) functions.
人类血小板是无核血细胞，可保留细胞质mRNA并维持功能完整的蛋白质翻译能力。我们采用了微阵列和基因表达系列分析（SAGE）的互补技术，对高度纯化的人类血小板进行基因谱分析。使用Affymetrix HG-U95Av2约12600个探针集的微阵列分析最大程度地鉴定出2147个（范围为13%-17%）血小板表达的转录本的表达，其中约22%共同参与代谢和受体/信号传导，且未分配功能的基因占比过高（32%）。相比之下，使用II型限制性内切酶MmeI（产生21个碱基对[bp]或22-bp标签）的改良SAGE方案表明，89%的标签代表线粒体（mt）转录本（富含16S和12S核糖体RNA），这可能与在没有核衍生转录本的情况下持续的mt转录有关。非mt SAGE标签的频率与通过微阵列分析确定的最“丰富”转录本的平均差异值（相对表达）平行，从而确定了两种血小板分析技术的一致性。定量逆转录-聚合酶链反应（PCR）证实了mt衍生转录本的最高频率，以及在最丰富的前5个转录本中神经颗粒素（NGN，一种蛋白激酶C底物）和补体溶解抑制剂clusterin的mRNA。为进行确证性表征，进行了免疫印迹和流式细胞术分析，证实了clusterin在细胞表面大量表达以及NGN在细胞内表达。这些观察结果表明高转录本丰度与蛋白质表达之间存在很强的相关性，并确立了转录本分析作为一种工具来鉴定可能调节正常和病理性血小板（和/或巨核细胞）功能的新型血小板蛋白的有效性。
