QuantiGene Plex assay

Description

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) .

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