QuantiGene Plex assay

Preliminary data for the selection of housekeeping genes suitable for our cells to be used as normaliser for the full experiment that will allow us to identify genes potentially modified by the infection with ZIKV and counteracted with the heparin treatment.
Instead of performing RNAseq analyses, we decided to opt for QuantiGene Plex (Thermofisher). This test incorporate branched DNA technology for accurate gene expression profiling. Branched DNA assays allow direct measurement of RNA transcripts using signal amplification (Luminex 200 technology) rather than target amplification.
We decided to include 4 conditions: uninfected, uninfected + heparin, ZIKV and ZIKV + heparin, and three time points: 4, 24, 48 h post infection, for a total of 12 samples. We performed three 1:4 serial dilutions of all samples. Then, we load the plate with all undiluted samples + all dilutions made. We performed the experiment according to the manufacturer’s instructions (Thermofisher). Thanks to the Thermofisher free online software, we performed the analyses of the data we obtained from the Luminex reading.
From the analyses file, we highlighted the data that were saturated (over 20000 reads), then we remove data from "uninfected + heparin 4h" and "ZIKV 4h" since the preparation was not perfect and the data were not reliable (incorrect serial dilution). We selected the samples in which genes are not saturated (dilution 1:64), and we performed the geometric mean (GEOMEAN) of all genes expressed by the same sample. We divided each values by its GEOMEAN and we calculated the standard deviation (SD). We selected the genes with the lower SD.
This experiment allowed us to identify the best 4 housekeeping genes: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK1), ubiquitin C (UBC) and the ATPase H+ transporting the A subunit V1 (ATP6V1A) .

本数据集旨在为筛选适用于本细胞系的内参基因提供初步数据，这些基因将被用于标准化整个实验，以便我们能够识别出可能受到寨卡病毒感染而受到肝素治疗的拮抗作用的基因。鉴于RNA测序分析的复杂性，我们决定采用Thermofisher公司的QuantiGene Plex技术。该测试集成了分支DNA技术，以实现精确的基因表达谱分析。分支DNA检测通过信号放大（Luminex 200技术）直接测量RNA转录本，而非目标扩增。我们选取了以下四种条件：未感染、未感染+肝素、寨卡病毒和寨卡病毒+肝素，以及三个时间点：感染后4小时、24小时、48小时，共计12个样本。对全部样本进行了三次1:4的系列稀释。随后，我们将所有未稀释样本及所有稀释样本加载至微孔板中。实验操作严格遵循制造商（Thermofisher）的指导说明。得益于Thermofisher免费在线软件，我们对其从Luminex读取的数据进行了分析。从分析文件中，我们筛选出饱和数据（读取数超过20000次），随后移除了“未感染+肝素4小时”和“寨卡病毒4小时”的数据，因为样本制备不完善，数据可靠性不高（稀释比例错误）。我们选择了基因未饱和的样本（稀释比例为1:64），并对同一样本表达的所有基因进行了几何平均（GEOMEAN）计算。我们将每个值除以其GEOMEAN，并计算标准差（SD）。我们选择了标准差较低的基因。该实验帮助我们确定了最佳的内参基因，包括甘油醛-3-磷酸脱氢酶（GAPDH）、磷酸甘油酸激酶1（PGK1）、泛素C（UBC）以及H+转运的ATPase V1的A亚基（ATP6V1A）。