少白细胞血小板miRNA提取方法

Method of extraction of miRNA from human leucocyte depleted-platelet

目的:建立常规梯度离心结合MACS磁珠分选纯化和小分子RNA分离试剂盒简便快速提取血小板miRNA的方法。方法:利用常规梯度离心获取血小板后,结合MACS磁珠分选和小分子RNA分离试剂盒提取少白细胞血小板(LDP)小分子RNA;经血小板计数、CD45和GPIIb mRNA marker PCR扩增检测,判定纯化效果;通过小分子RNA浓度、纯度及毛细管电泳完整性检测,以及内参U6和血小板miR-449b加尾及引物延伸RT-PCR后PCR扩增结果验证构建血小板miRNA的cDNA模板真实性。结果:提取小分子RNA纯度较高,有效去除白细胞污染,虽然含量较少,但内参U6和血小板miR-449b真实性验证结果提示,构建血小板miRNA的cDNA模板质量较高。结论:常规梯度离心结合MACS磁珠分选纯化和小分子RNA分离试剂盒,可有效去除白细胞污染,保证小分子RNA完整性,从而提取高质量血小板miRNA的cDNA模板,可用于后续血小板miRNA试验研究。

Objective： To establish a simple and convenient method isolation of high-quality miRNA from human leukocyte-depleted platelet preparation. Methods： Small RNA in healthy platelets was extracted by conventional gradient centrifugation, MACS and the mirVanaTM miRNA Isolation Kit. Leukocyte contamination in the final purified platelet preparation was estimated by conventional gradient centrifugation, platelet counting and the transcript levels of the gene encoding CD45/GPIIb marker in platelet preparations. The concentration, purity and integrity were detected, and a miRNA cDNA library which was constructed by RNA-tailing and primer-extension reverse transcription （RT）-PCR was used as the template for the amplification of U6 and miR-449b to verify the authenticity. Results： Small RNA was extracted with high purity, high efficiency of leukocyte depletion, Its cDNA had high authenticity despite its low concentration. Conclusion： A method of extraction of microRNA from human platelet preparation with high efficiency of leukocyte depletion, and high quality of miRNA＇ eDNA library is constructed, which is applicative to future miRNA experiments.