Transcript profiling of human platelets using microarray and serial analysis of gene expression (SAGE).
Platelets are anucleated cells that are generated from megakaryocytes via thrombopoiesis. They lack genomic DNA but have a pool of individual mRNA transcripts. Taken together, these mRNAs constitute a platelet transcriptome. Platelets have a unique and reproducible transcript profile, which includes approximately 1,600-3,000 individual transcripts. In this chapter, we will focus on platelet purification and on transcript profiling using an Affymetrix microarray platform and serial analysis of gene expression (SAGE). Platelet purification is described in detail. Large-scale platelet purification schema is designed to purify platelets from apheresis platelet bags (approximately 3-5 x 10(11) platelets/bag). Modification of this schema --small-scale platelet purification--is designed to isolate platelets from 20 ml of peripheral blood. This chapter provides detailed protocols for microarray and SAGE transcript profiling. We also discuss peculiarities of platelet purification, RNA isolation, and transcript profiling.
Division of Hematology/Oncology, Department of Medicine, State University of New York at Stony Brook, Stony Brook, NY, USA.
Dmitri V
DV
Gnatenko
John J
JJ
Dunn
John
J
Schwedes
Wadie F
WF
Bahou
D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010983:Plateletpheresis / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D012333:RNA, Messenger