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 ∼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 (∼3–5 × 1011 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.
Key WordsPlatelet RNA microarray serial analysis of gene expression
This work was supported by NIH/NHLBI grants R21 HL076457 (D.V.G.) and HL086376 (W.F.B); Department of Defense grant MP048005 and a Targeted Research Award (D.V.G.) from Stony Brook University. Studies at the Brookhaven National Laboratory were supported by a Laboratory Directed Research and Development award (J.J.D.) and by the Offices of Biological and Environmental Research, and of Basic Energy Sciences (Division of Energy Biosciences) of the US Department of Energy.
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