RNase H2 依赖性聚合酶链反应以及在样本采集、储存和分析过程中消除混杂因素，增强了牛初乳中的 microRNAs 在人体中具有生物利用度的证据。
RNase H2-Dependent Polymerase Chain Reaction and Elimination of Confounders in Sample Collection, Storage, and Analysis Strengthen Evidence That microRNAs in Bovine Milk Are Bioavailable in Humans.
机构信息
Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE.
出版信息
J Nutr. 2018 Jan 1;148(1):153-159. doi: 10.1093/jn/nxx024.
BACKGROUND
Evidence suggests that dietary microRNAs (miRs) are bioavailable and regulate gene expression across species boundaries. Concerns were raised that the detection of dietary miRs in plasma might have been due to sample contamination or lack of assay specificity. Objectives: The objectives of this study were to assess potential confounders of plasma miR analysis and to detect miRs from bovine milk in human plasma.
METHODS
Potential confounders of plasma miR analysis (circadian rhythm, sample collection and storage, calibration, and erythrocyte hemolysis) were assessed by quantitative reverse transcriptase polymerase chain reaction (PCR) by using blood from healthy adults (7 men, 6 women; aged 23-57 y). Bovine miRs were analyzed by RNase H2-dependent PCR (rhPCR) in plasma collected from a subcohort of 11 participants before and 6 h after consumption of 1.0 L of 1%-fat bovine milk.
RESULTS
The use of heparin tubes for blood collection resulted in a complete loss of miRs. Circadian variations did not affect the concentrations of 8 select miRs. Erythrocyte hemolysis caused artifacts for some miRs if plasma absorbance at 414 nm was >0.300. The stability of plasma miRs depended greatly on the matrix in which the miRs were stored and whether the plasma was frozen before analysis. Purified miR-16, miR-200c, and cel-miR-39 were stable for ≤24 h at room temperature, whereas losses equaled ≤80% if plasma was frozen, thawed, and stored at room temperature for as little as 4 h. rhPCR distinguished between bovine and human miRs with small variations in the nucleotide sequence; plasma concentrations of Bos taurus (bta)-miR-21-5p and bta-miR-30a-5p were >100% higher 6 h after milk consumption than before milk consumption.
CONCLUSIONS
Confounders in plasma miR analysis include the use of heparin tubes, erythrocyte hemolysis, and storage of thawed plasma at room temperature. rhPCR is a useful tool to detect dietary miRs.
背景
有证据表明，膳食 microRNAs（miRs）是有生物活性的，可以在物种间调节基因表达。人们担心在血浆中检测到膳食 miR 可能是由于样品污染或缺乏检测特异性所致。目的：本研究旨在评估血浆 miR 分析的潜在混杂因素，并检测人血浆中的牛源乳 miR。
方法
通过定量逆转录聚合酶链反应（PCR），利用健康成年人（7 名男性，6 名女性；年龄 23-57 岁）的血液评估血浆 miR 分析的潜在混杂因素（昼夜节律、样品采集和储存、校准和红细胞溶血）。采用依赖 RNase H2 的 PCR（rhPCR）分析来自 11 名参与者亚组的血浆，这些参与者在饮用 1.0 L 1%-脂肪牛乳前后 6 h 内采集血浆。
结果
使用肝素管采集血液会导致 miR 完全丢失。昼夜节律变化不会影响 8 种选定 miR 的浓度。如果血浆在 414 nm 处的吸光度 >0.300，则红细胞溶血会导致某些 miR 出现假象。血浆 miR 的稳定性在很大程度上取决于储存 miR 的基质以及分析前是否冷冻血浆。在室温下，纯化的 miR-16、miR-200c 和 cel-miR-39 可稳定 ≤24 h，而如果将血浆冷冻、解冻并在室温下储存≤4 h，则损失可达 ≤80%。rhPCR 可以区分牛源和人源 miR，核苷酸序列略有差异；与饮用牛乳前相比，饮用牛乳后 6 h，牛源 miR-21-5p 和 miR-30a-5p 的血浆浓度高出 >100%。
结论
血浆 miR 分析的混杂因素包括使用肝素管、红细胞溶血以及解冻后在室温下储存血浆。rhPCR 是一种有用的工具，可用于检测膳食 miR。
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