Recovery of cell‐free mRNA and microRNA from human semen based on their physical nature
Recovery of cell‐free mRNA and microRNA from human semen based on their physical nature
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DOI：
10.1002/bab.1172
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发表时间：
2014-05
影响因子：
2.8
通讯作者：
Liping Wang;Jinming Lv;Cuicui Guo;Honggang Li;Chengliang Xiong
中科院分区：
文献类型：
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作者：
Liping Wang;Jinming Lv;Cuicui Guo;Honggang Li;Chengliang Xiong
在人类精液中发现了无细胞精液 mRNA (cfs-mRNA) 和 microRNA (cfs-miRNA)，并被报道为有前途的男性生殖器官疾病和法医鉴定的非侵入性生物标志物。然而，精浆由于其成分复杂且蛋白质、DNA 和多糖含量高，对于 RNA 提取尤其具有挑战性。在这里，我们基于我们之前对 cfs-mRNA 和 cfs-miRNA 物理性质的发现，报告了一种新颖、简单且可靠的方法，用于从人类精液中分离 cfs-mRNA 和 cfs-miRNA。精液微泡（直径 0.1-0.5 µm）含有大部分 cfs-mRNA，通过微过滤器富集。大多数 cfs-miRNA 结合的蛋白质复合物通过超滤器进行富集。收获含有RNA的复合物或微泡，避免了人类精液中其他成分的影响，从而有利于RNA的分离和纯化。这种新方法可以根据cfs-mRNA和cfs-miRNA的物理性质，有效地分别分离cfs-mRNA和cfs-miRNA，且RNA纯度高，DNA污染低。它还可以应用或修改以分离其他液体中的无细胞 RNA。
Cell‐free seminal mRNA (cfs‐mRNA) and microRNA (cfs‐miRNA) have been found in human ejaculate and reported as promising noninvasive biomarkers for disorders of male reproductive organs and forensic identification. However, seminal plasma is particularly challenging for RNA extraction due to its complicated composition and high content of protein, DNA, and polysaccharide. Here, we report a novel, simple, and reliable method for the isolation of cfs‐mRNA and cfs‐miRNA from human semen based on our previous findings of their physical nature. Seminal microvesicles (0.1–0.5 µm in diameter), which contain the majority of cfs‐mRNA, were enriched by a microfilter. Protein complexes, which most cfs‐miRNA is bound with, were enriched by an ultrafilter. Harvesting the complexes or microvesicles, in which RNAs exist, avoided the influence of other components in human semen, thus favoring RNA isolation and purification. This new method can efficiently isolate cfs‐mRNA and cfs‐miRNA separately based on their physical nature, with high RNA purity, and low DNA contamination. It may also be applied or modified to isolate cell‐free RNAs in other fluids.