Fluidigm real time pcr analysis software 4

Initially, the collected samples were screened for IAV by using the high-throughput rtPCR platform BioMark (Fluidigm, South San Francisco, USA) and the 192.24 Dynamic array (DA) integrated fluidic circuit (IFC) chip (Fluidigm). A 4 µL sample mix was prepared by mixing 2.2 µL of pre-sample mix (prepared by mixing 2 µL of 2X TaqMan Gene Expression Mastermix (Applied Biosystem) and 0.2 µL of 20X sample loading reagent (Fluidigm) for each sample) and 1.8 µL of pre-amplified sample. Similarly, 2 µL primer/probe stock was mixed with 2 µL of 2X assay loading reagent (Fluidigm). Three µL of the assay mix and 3 µL of sample mix was loaded into the respective inlets of the 192.24DA IFC chip. The 192.24 DA IFC chip was placed in the IFC controller RX for loading and mixing for approximately 30 min. Finally, the chip was inserted into the high-throughput rtPCR platform BioMark (Fluidigm) for thermal cycling with the following cycling condition: 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. All samples were tested in duplicates. Positive and non-template (nuclease-free water) controls were included. Amplification curves and cycle threshold (Ct) values were obtained on the BioMark system and finally analysed using Fluidigm Real Time PCR Analysis software 4.1.3 (Fluidigm) as previously described [41 (link)].

Gene expression analyses were performed as previously described (35 (link)). Briefly, data were collected with the Fluidigm Real-Time PCR Analysis software 4.1.3 (Fluidigm Corporation, USA) and analyzed with the DAG expression software 1.0.5.6 (37 (link)). The relative standard curve method (see Applied Biosystems user bulletin #2) was applied to compare gene expression levels of LN cells cultured in different conditions against those of freshly prepared LN cells, using multiple reference gene normalization (GAPDH, HPRT1 and UbC). Relative expression of IFN-λ1 and IFN-λ3 was calculated according to the 2^-ΔΔCT method (38 (link)), using the same normalizer genes, since expression levels of these genes in control samples were too low to generate standard curves. Relative expression of each gene in a particular sample was expressed in mean fold-change values (Fc) and are shown in Supplementary Table 1.
The unpaired t-test was used to statistically compare the relative expression levels of genes from LN cells exposed to MERS-CoV Qatar15/2015, Egypt/2013 and those of cells cultured in media only. All statistical analyses were performed using GraphPad Prism 9.3.1 (GraphPad Software, USA). Differences were considered significant at p-values < 0.05.