Size-Exclusion Chromatography Separation Reveals That Vesicular and Non-Vesicular Small RNA Profiles Differ in Cell Free Urine.
Size-Exclusion Chromatography Separation Reveals That Vesicular and Non-Vesicular Small RNA Profiles Differ in Cell Free Urine.
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DOI：
10.3390/ijms22094881
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发表时间：
2021-05-05
影响因子：
5.6
通讯作者：
Williams TL
中科院分区：
文献类型：
--
作者：
Karttunen J;Stewart SE;Kalmar L;Grant AJ;Karet Frankl FE;Williams TL
尿细胞外囊泡（EV）及其RNA货物是各种疾病的生物标志物的新来源。我们的目的是确定在小RNA分析之前从人尿液中分离小（<200 nm）EV的最佳方法。使用三种方法浓缩来自过滤的健康志愿者尿液的EV：超离心（UC）;基于沉淀的试剂盒（PR）;和超滤（UF）。通过尺寸排阻色谱法（SEC）进一步纯化EV。用透射电子显微镜（TEM）、蛋白质印迹、纳米颗粒跟踪分析（NTA）和Agilent Bioanalyzer Small RNA试剂盒分析EV制备物。UF在SEC之前和之后都产生最高数量的颗粒。来自UF浓缩尿液的小RNA分析鉴定了10-40个核苷酸（nt）和40-80个核苷酸处的两个主峰。相比之下，在UC、PR或SEC与任何浓缩方法组合后获得的EV制剂主要含有40-80 nt大小的小RNA。来自UF+SEC的蛋白质级分含有大小为10-40 nt的小RNA（与miRNA一致）。这些数据表明，过滤尿液中大多数microRNA大小的RNA与小尺寸EV无关，并强调了在小RNA分析之前从尿液EV制剂中去除非囊泡蛋白和RNA的重要性。
Urinary extracellular vesicles (EVs) and their RNA cargo are a novel source of biomarkers for various diseases. We aimed to identify the optimal method for isolating small (<200 nm) EVs from human urine prior to small RNA analysis. EVs from filtered healthy volunteer urine were concentrated using three methods: ultracentrifugation (UC); a precipitation-based kit (PR); and ultrafiltration (UF). EVs were further purified by size-exclusion chromatography (SEC). EV preparations were analysed with transmission electron microscopy (TEM), Western blotting, nanoparticle tracking analysis (NTA) and an Agilent Bioanalyzer Small RNA kit. UF yielded the highest number of particles both before and after SEC. Small RNA analysis from UF-concentrated urine identified two major peaks at 10–40 nucleotides (nt) and 40–80 nt. In contrast, EV preparations obtained after UC, PR or SEC combined with any concentrating method, contained predominantly 40–80 nt sized small RNA. Protein fractions from UF+SEC contained small RNA of 10–40 nt in size (consistent with miRNAs). These data indicate that most of the microRNA-sized RNAs in filtered urine are not associated with small-sized EVs, and highlights the importance of removing non-vesicular proteins and RNA from urine EV preparations prior to small RNA analysis.
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DOI：
10.1007/s00018-018-2773-4
发表时间：
2018-08
期刊：
Cellular and molecular life sciences : CMLS
影响因子：
--
作者：
Karimi N;Cvjetkovic A;Jang SC;Crescitelli R;Hosseinpour Feizi MA;Nieuwland R;Lötvall J;Lässer C
通讯作者：
Lässer C
影响因子：
5.5
作者：
Marchand V;Pichot F;Thüring K;Ayadi L;Freund I;Dalpke A;Helm M;Motorin Y
通讯作者：
Motorin Y
影响因子：
4.6
作者：
Gheinani AH;Vögeli M;Baumgartner U;Vassella E;Draeger A;Burkhard FC;Monastyrskaya K
通讯作者：
Monastyrskaya K
影响因子：
4.6
作者：
Brennan, K.;Martin, K.;Mc Gee, M. M.
通讯作者：
Mc Gee, M. M.
影响因子：
4.1
作者：
Armstrong, David A.;Dessaint, John A.;Ashare, Alix
通讯作者：
Ashare, Alix