Crossland Rachel E, Norden Jean, Bibby Louis A, Davis Joanna, Dickinson Anne M
Institute of Cellular Medicine, Medical School, Framlington Place, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.
Institute of Cellular Medicine, Medical School, Framlington Place, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.
J Immunol Methods. 2016 Feb;429:39-49. doi: 10.1016/j.jim.2015.12.011. Epub 2015 Dec 23.
MicroRNAs are small regulatory molecules that demonstrate useful biomarker potential. They have been recognised in biofluids, where they are protected from degradation by encapsulation into extracellular vesicles (EVs). A number of commercial products are available for the isolation of EVs and their RNA content; however, extensive protocol comparisons are lacking. Furthermore, robust qRT-PCR assessment of microRNA expression within EVs is problematic, as endogenous controls (ECs) previously used in cellular samples may not be present. This study compares EV isolation and RNA extraction methods (EV precipitation reagents, RNA isolation kits and ultracentrifugation) from serum or urine samples and evaluates suitable ECs for incorporation into qRT-PCR analysis. Results were assessed by electron microscopy, nanoparticle tracking analysis and bioanalyzer concentrations. The stability of 8 ECs was compared for both serum and urine EV RNA and retrospectively validated in independent cohorts (serum n=55, urine n=50). The Life Technologies precipitation reagent gave superior serum EV recovery compared to SBI reagent, as assessed by NTA size distribution, increased RNA concentration, and lower small RNA Ct values. Similarly, the Norgen Biotek Urine Exosome RNA Isolation Kit gave improved results for urine EV isolation compared to ultracentrifugation, when determined by the same parameters. The Qiagen miRNeasy™ RNA isolation kit gave suitable serum EV RNA concentrations compared to other kits, as assessed by Bioanalyzer and small RNA qRT-PCR. Small RNAs HY3 (S.D=1.77, CoV=6.2%) and U6 (S.D=2.14, CoV=8.6%) were selected as optimal ECs for serum EV microRNA expression analysis, while HY3 (S.D=1.67, CoV=6.5%) and RNU48 (S.D=1.85, CoV=5.3%) were identified as suitable for urine studies. In conclusion, this study identifies optimal methods for isolation of serum and urine EV RNA, and suitable ECs for normalisation of qRT-PCR studies. Such reports should aid in the standardisation of EV microRNA data, particularly for biomarker studies.
微小RNA是一类具有潜在生物标志物价值的小型调控分子。它们在生物流体中被发现，通过被包裹进细胞外囊泡（EV）而免受降解。市面上有多种用于分离EV及其RNA成分的商业产品；然而，目前缺乏广泛的方案比较。此外，对EV内微小RNA表达进行可靠的定量逆转录聚合酶链反应（qRT-PCR）评估存在问题，因为之前在细胞样本中使用的内参（EC）在EV中可能不存在。本研究比较了从血清或尿液样本中分离EV和提取RNA的方法（EV沉淀试剂、RNA提取试剂盒和超速离心法），并评估了适用于纳入qRT-PCR分析的EC。通过电子显微镜、纳米颗粒跟踪分析和生物分析仪浓度来评估结果。比较了8种EC在血清和尿液EV RNA中的稳定性，并在独立队列中进行回顾性验证（血清n = 55，尿液n = 50）。通过纳米颗粒跟踪分析（NTA）大小分布、RNA浓度增加和小RNA的Ct值降低评估，与SBI试剂相比，赛默飞世尔科技的沉淀试剂能更好地回收血清EV。同样，当通过相同参数测定时，与超速离心法相比，诺金生物科技的尿液外泌体RNA分离试剂盒在尿液EV分离方面效果更佳。通过生物分析仪和小RNA qRT-PCR评估，与其他试剂盒相比，Qiagen miRNeasy™ RNA分离试剂盒能获得合适的血清EV RNA浓度。小RNA HY3（标准差 = 1.77，变异系数 = 6.2%）和U6（标准差 = 2.14，变异系数 = 8.6%）被选为血清EV微小RNA表达分析的最佳EC，而HY3（标准差 = 1.67，变异系数 = 6.5%）和RNU48（标准差 = 1.85，变异系数 = 5.3%）被确定适用于尿液研究。总之，本研究确定了分离血清和尿液EV RNA的最佳方法，以及用于qRT-PCR研究标准化的合适EC。此类报告应有助于EV微小RNA数据的标准化，特别是对于生物标志物研究。
