Real time pcr analysis software v 4.1.2

A total of 90-target genes, relevant to monocyte function, that included major cytokines and pathway proteins involved in inflammation were selected for pre-amplification in the IFCs using the Fluidigm C1 Single-Cell Auto Prep System according to the manufacturer’s protocol. qPCR-based gene expression assay of the target gene pre-amplified cDNAs were carried out using 96.96 IFCs on the BioMark HD System (Fluidigm) as described in the protocol. Raw data was analyzed using the Fluidigm Real-Time PCR Analysis software (v. 4.1.2) and quality check was performed by inspecting melt curves, amplification curves. A failure score was calculated for each cell as described previously [9 (link), 13 (link)]. Cells with failure score (total CT value) greater than two standard deviations above the mean were excluded from downstream analysis. The limit of detection CT values was set at 28 [10 (link)]; CT values greater than or equal to 28 were considered non-detected and were assigned a value of zero for analysis. Gene expression values were calculated by subtracting the threshold cycle value for each gene for each cell from the number of cycles in the PCR reaction. In this way, higher numbers represent greater gene expression, and lower numbers indicate less expression.

Preamplified samples (diluted 1:5 with H₂O) and TaqMan 20x assays miRNAs were applied to primed 96.96 dynamic array chips using loading and assay reagents according to the manufacturer (Fluidigm Corp., USA). All miR-assays were performed in duplicate. The samples of this study were analyzed on three 96x96 chips (Fluidigm Corp., USA) with two different 48-miRNA assay panels (for a total of 6 chips). After loading the reaction chambers using the integrated fluid circuit (IFC) HX controller from Fluidigm, the real-time PCR including image capture after each cycle was performed in a BioMark real-time PCR system (Fluidigm Corp., USA) using single probe (FAM-MGB, reference: ROX) settings and GE 96x96 standard v1 protocol with 40 cycles. Data processing took place using the Fluidigm real-time PCR analysis software (v. 4.1.2).