Transcript profiling of human platelets using microarray and serial analysis of gene expression.
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.
Gnatenko
Dmitri V
DV
Department of Medicine, Program in Genetics, State University of New York, Stony Brook 11794-8151, USA.
Dunn
John J
JJ
McCorkle
Sean R
SR
Weissmann
David
D
Perrotta
Peter L
PL
Bahou
Wadie F
WF
D001483:Base Sequence; D001792:Blood Platelets / Q000737:chemistry* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000097:blood / Q000235:genetics; D002469:Cell Separation; D051152:Clusterin; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D020869:Gene Expression Profiling; D015723:Gene Library; D006023:Glycoproteins / Q000097:blood / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000737:chemistry; D018832:Molecular Chaperones / Q000097:blood / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000097:blood / Q000235:genetics; D051579:Neurogranin; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C495242:CLU protein, human; D002148:Calmodulin-Binding Proteins; D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; C496649:NRGN protein, human; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D051579:Neurogranin; C049760:endodeoxyribonuclease MmeI; D015252:Deoxyribonucleases, Type II Site-Specific