Pre-Analytical Handling Conditions and Small RNA Recovery from Urine for miRNA Profiling
Pre-Analytical Handling Conditions and Small RNA Recovery from Urine for miRNA Profiling
复制标题
DOI：
10.1016/j.jmoldx.2018.04.003
复制
发表时间：
2018-09-01
影响因子：
4.1
通讯作者：
Ashare, Alix
中科院分区：
文献类型：
--
作者：
Armstrong, David A.;Dessaint, John A.;Ashare, Alix
目前尚无用于尿液分析前处理的标准化方案，以最好地保存用于 miRNA 分析研究的小 RNA。 miRNA 作为潜在生物标志物是一个有吸引力的候选者，因为它在体液中具有高度稳定性，并且能够在多个高通量平台上进行定量。我们比较了不同分析前处理条件下尿液中小 RNA 的回收率和稳定性，并就哪些条件优化无细胞尿液和尿液细胞外囊泡 (EV) 中 miRNA 的产量提出了建议。使用亲和浆液从尿液中分离小 RNA，我们发现尿液样本在处理前在室温（20 摄氏度）下保存长达 8 小时，可从 EV 中获得最高量的完整小 RNA。当在 EV 分离之前保持 2°C 至 4°C 和/或冷冻时，一些 miRNA 会从尿液样本中丢失，这可能是因为 EV 被困在尿调节素沉淀物中。然而，我们发现，将含有冷诱导沉淀的尿液在 37 摄氏度下简单孵育 5 分钟，可以重新溶解大部分沉淀，从而提高 EV 和 miRNA 的回收率。最后，当全尿在 37 摄氏度下保存短短 4 小时时，小 RNA 的完整性可能会受到损害，并且不利于有效的 miRNA 分析。
There are currently no standardized protocols for pre-analytical handling of urine to best preserve small RNA for miRNA profiling studies. miRNA is an attractive candidate as a potential biomarker because of the high level of stability in body fluids and its ability to be quantified on multiple high-throughput platforms. We present a comparison of small RNA recovery and stability in urine under alternate pre-analytical handling conditions and extend recommendations on what conditions optimize yield of miRNA from cell-free urine and urine extracellular vesicles (EVs). Using an affinity slurry for isolation of small RNA from urine, we found that urine samples held at room temperature (20 degrees C) for up to 8 hours before processing yield the highest amounts of intact small RNAs from EVs. Some miRNA is lost from urine samples when held 2 degrees C to 4 degrees C and/or frozen before EV isolation, likely because of EV entrapment in uromodulin precipitates. However, we found that a simple 5-minute incubation of urine containing cold-induced precipitate at 37 degrees C resolubilizes much of this precipitate and results in an increased recovery of EVs and miRNAs. Finally, small RNA integrity can be compromised when whole urine is held at 37 degrees C for as little as 4 hours and is not conducive to efficient miRNA profiling.