epigenetic_qPCR_data_final.xlsx

We analysed whole blood and nasopharyngeal swabs from COVID-19 patients in two different cohorts collected at hospitals in Germany (Bochum) and Spain (Valencia) by epigenetic immune cell quantification using qPCR assays (demethyl-specific). The aim was to investigate the prognostic potential of this approach to identify patients with higher risk for a poor outcome. Also, we compared epigenetic data of patients with those of healthy donors. <br> Dataset includes Cp (crossing-point) values, cell specific plasmid units (PU) per qPCR reaction resulting from epigenetic qPCR analyses (calculated based on a quantification standard), as well as coefficient of variation (C.V.). PUs were translated into immune cell counts relative to the housekeeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) for each sample (peripheral whole blood, dried blood spots and swabs). GAPDH is also targeted by an epigenetic qPCR assay to quantify all GAPDH PUs. <br> All samples were lysed to extract genomic DNA. Afterwards, DNA was incubated with ammonium-bisulfite to convert all unmethylated CpGs (cytosin-guanine-dinucleotide) to uracil residues by deamination and desulphonation reaction without alteration of methylated CpGs. Within the subsequent epigenetic qPCR generated uracils are changed to thymine by proof-reading capability of the used DNA-Polymerase. <br> The epigenetic qPCR assays consist of demethyl-specific primer pairs and probes to detect cell type-specific demethylation within genes of interest. <br>

本研究针对采自德国波鸿（Bochum）与西班牙巴伦西亚（Valencia）两地医院的两个不同队列的COVID-19患者，采用去甲基特异性qPCR（quantitative polymerase chain reaction，实时荧光定量聚合酶链反应）检测开展表观遗传免疫细胞定量分析。本研究旨在探究该方法识别不良预后高风险患者的预后价值，同时对比了COVID-19患者与健康供者的表观遗传数据。
本数据集包含Cp（crossing-point，循环阈值）值、基于定量标准计算得到的表观遗传qPCR分析中每次qPCR反应的细胞特异性质粒单位（plasmid units, PU），以及变异系数（coefficient of variation, C.V.）。针对每一类样本（外周全血、干血斑及鼻咽拭子），研究人员将PU值转换为相对于持家基因GAPDH（glyceraldehyde 3-phosphate dehydrogenase，甘油醛-3-磷酸脱氢酶）的免疫细胞计数。同时，表观遗传qPCR检测也会靶向GAPDH以定量所有GAPDH相关PU值。
所有样本均经裂解处理以提取基因组DNA。随后，将DNA与亚硫酸氢铵（ammonium-bisulfite）孵育，通过脱氨基与脱硫反应将所有未甲基化的CpG（cytosin-guanine-dinucleotide，胞嘧啶-鸟嘌呤二核苷酸）转化为尿嘧啶残基，而甲基化CpG则保持不变。
在后续的表观遗传qPCR反应中，所用DNA聚合酶（DNA-Polymerase）的校对活性会将反应中生成的尿嘧啶转换为胸腺嘧啶。
该表观遗传qPCR检测体系包含去甲基特异性引物对与探针，用于检测目标基因内的细胞类型特异性去甲基化修饰。