Transcript profiling of human platelets using microarray and serial analysis of gene expression - PubMed (original) (raw)
. 2003 Mar 15;101(6):2285-93.
doi: 10.1182/blood-2002-09-2797. Epub 2002 Nov 14.
Affiliations
- PMID: 12433680
- DOI: 10.1182/blood-2002-09-2797
Free article
Transcript profiling of human platelets using microarray and serial analysis of gene expression
Dmitri V Gnatenko et al. Blood. 2003.
Free article
Abstract
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.
Comment in
- Evaluating the relevance of the platelet transcriptome.
Weyrich AS, Zimmerman GA. Weyrich AS, et al. Blood. 2003 Aug 15;102(4):1550-1. doi: 10.1182/blood-2003-05-1429. Blood. 2003. PMID: 12900352 No abstract available.
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