Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients.
Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients.
复制标题
DOI：
10.1371/journal.pone.0183435
复制
发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Endlich N
中科院分区：
文献类型：
--
作者：
Lange T;Stracke S;Rettig R;Lendeckel U;Kuhn J;Schlüter R;Rippe V;Endlich K;Endlich N
慢性肾脏病（CKD）是一种严重的疾病，在全球范围内发病率不断增加。早期发现可能有助于防止其进展，并将心血管疾病作为主要合并症之一的风险降至最低。最近，细胞外miRNA如尿外泌体miRNA作为可以通过RT-qPCR测定的非侵入性生物标志物变得非常有趣。但到目前为止，关于从体液中分离的miRNAs的正常化还没有达成共识。本研究分析了miRNAs miR-16、miR-92 a、miR-21、miR-124 a和小核RNA RNU 6 B作为从CKD患者分离的外泌体miRNAs表达研究中的内源性参考基因的适用性。为此，在从33名CKD患者和5名健康对照中分离尿外泌体后，通过RT-qPCR测定miRNA表达水平。表达数据用标准化测定软件Normalware、BestKeeper、GeNorm和DeltaCt分析。我们的结果显示，在尿液外泌体中有四种候选miRNA的丰富表达，并且没有可检测到的RNU 6 B表达。我们确定miR-16是我们数据集中最稳定的内源性参考基因，使其成为CKD患者尿外泌体miRNA研究的合适内源性参考基因。
Chronic kidney disease (CKD) is a severe disorder with an increasing incidence worldwide. An early detection may help to prevent its progression and to minimize the risk of cardiovascular diseases as one of the major comorbidities. Recently, extracellular miRNAs like urinary exosomal miRNAs became of great interest as non-invasive biomarkers which can be determined by RT-qPCR. But until now, there is no consensus regarding the normalization of miRNAs isolated from body fluids. The present study analyzed the miRNAs miR-16, miR-92a, miR-21, miR-124a and the small nuclear RNA RNU6B for their applicability as an endogenous reference gene in expression studies of exosomal miRNAs isolated from CKD patients. For this purpose, miRNA expression levels were determined by RT-qPCR after the isolation of urinary exosomes from 33 CKD patients and from 5 healthy controls. Expression data was analyzed with the normalization determination software NormFinder, BestKeeper, GeNorm and DeltaCt. Our results revealed an abundant expression of the four candidate miRNAs in urinary exosomes and no detectable expression of RNU6B. We identified miR-16 as the most stable endogenous reference gene in our data set, making it a suitable endogenous reference gene for miRNA studies of urinary exosomes derived from CKD patients.