Translational Research in Respiratory Medicine, University Hospital Arnau de Vilanova and Santa Maria, IRBLleida, Lleida, Spain; CIBER of Respiratory Diseases (CIBERES), Institute of Health Carlos III, Madrid, Spain.
Translational Research in Respiratory Medicine, University Hospital Arnau de Vilanova and Santa Maria, IRBLleida, Lleida, Spain.
Int J Biol Macromol. 2024 Jun;269(Pt 2):131926. doi: 10.1016/j.ijbiomac.2024.131926. Epub 2024 Apr 28.
Circulating cell-free microRNAs (miRNAs) are promising biomarkers for medical decision-making. Suitable endogenous controls are essential to ensure reproducibility. We aimed to identify and validate endogenous reference miRNAs for qPCR data normalization in samples from SARS-CoV-2-infected hospitalized patients. We used plasma samples (n = 170) from COVID-19 patients collected at hospital admission (COVID-Ponent project, www.clinicaltrials.gov/NCT04824677). First, 179 miRNAs were profiled using RT-qPCR. After stability assessment, candidates were validated using the same methodology. miRNA stability was analyzed using the geNorm, NormFinder and BestKeeper algorithms. Stability was further evaluated using an RNA-seq dataset derived from COVID-19 hospitalized patients, along with plasma samples from patients with critical COVID-19 profiled using RT-qPCR. In the screening phase, after strict control of expression levels, stability assessment selected eleven candidates (miR-17-5p, miR-20a-5p, miR-30e-5p, miR-106a-5p, miR-151a-5p, miR-185-5p, miR-191-5p, miR-423-3p, miR-425-5p, miR-484 and miR-625-5p). In the validation phase, all algorithms identified miR-106a-5p and miR-484 as top endogenous controls. No association was observed between these miRNAs and clinical or sociodemographic characteristics. Both miRNAs were stably detected and showed low variability in the additional analyses. In conclusion, a 2-miRNA panel composed of miR-106a-5p and miR-484 constitutes a first-line normalizer for miRNA-based biomarker development using qPCR in hospitalized patients infected with SARS-CoV-2.
循环细胞游离 microRNAs（miRNAs）是用于医疗决策的有前途的生物标志物。合适的内参对于确保重现性至关重要。我们旨在鉴定和验证用于 SARS-CoV-2 感染住院患者样本 qPCR 数据归一化的内源性参考 miRNAs。我们使用了来自 COVID-19 患者入院时采集的血浆样本（COVID-Ponent 项目，www.clinicaltrials.gov/NCT04824677）（n=170）。首先，使用 RT-qPCR 对 179 个 miRNAs 进行了分析。在稳定性评估后，使用相同的方法对候选物进行了验证。使用 geNorm、NormFinder 和 BestKeeper 算法分析了 miRNA 的稳定性。使用来自 COVID-19 住院患者的 RNA-seq 数据集以及使用 RT-qPCR 分析的患有严重 COVID-19 的患者的血浆样本进一步评估了稳定性。在筛选阶段，在严格控制表达水平后，稳定性评估选择了 11 个候选物（miR-17-5p、miR-20a-5p、miR-30e-5p、miR-106a-5p、miR-151a-5p、miR-185-5p、miR-191-5p、miR-423-3p、miR-425-5p、miR-484 和 miR-625-5p）。在验证阶段，所有算法均将 miR-106a-5p 和 miR-484 鉴定为顶级内参。这些 miRNA 与临床或社会人口统计学特征之间没有观察到相关性。在额外的分析中，这两种 miRNA 均稳定检测到，且变异性低。总之，由 miR-106a-5p 和 miR-484 组成的 2-miRNA 面板构成了使用 qPCR 在感染 SARS-CoV-2 的住院患者中开发基于 miRNA 的生物标志物的一线标准化器。
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