A Rapid and Efficient Platelet Purification Protocol for Platelet Gene Expression Studies
A Rapid and Efficient Platelet Purification Protocol for Platelet Gene Expression Studies
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DOI：
10.1007/978-1-61779-307-3_12
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发表时间：
2012-01-01
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影响因子：
--
通讯作者：
Amisten, Stefan
中科院分区：
文献类型：
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作者：
Amisten, Stefan
从全血中分离纯化血小板是研究血小板基因表达的关键。为了成功地从全血中分离血小板，需要克服的主要障碍包括：（1）血小板活化;（2）白细胞和红细胞污染;以及（3）时间依赖性血小板mRNA降解。本章描述了一种从少量全血中分离人循环血小板的快速高效方法，该方法基于有效抑制血小板活化和过滤去除白细胞，然后磁珠去除残留的污染白细胞和红细胞。还描述了使用定量实时PCR和微阵列进行RNA提取、cDNA合成和血小板基因表达研究的方法。
Isolation of pure platelet samples from whole blood is crucial for the study of platelet gene expression. The main obstacles to overcome in order to successfully isolate platelets from whole blood include (1) platelet activation; (2) leukocyte and red blood cell contamination, and (3) time-dependent platelet mRNA degradation. This chapter describes a rapid and highly efficient method for isolating human circulating platelets from small volumes of whole blood based on efficient inhibition of platelet activation and leukocyte removal by filtration followed by magnetic bead-depletion of residual contaminating leukocytes and red blood cells. Also described are methods for RNA extraction, cDNA synthesis, and platelet gene expression studies using both quantitative real-time PCR and microarray.