Biomark qpcr platform

Microfluidic QPCR was performed on the 96.96 dynamic array (Fluidigm) using 2.25 µl of the diluted cDNA sample from the specific target amplification, along with Taqman assays for each gene, Taqman Universal Master Mix, and Fluidigm loading reagent as outlined in the manufacturer's protocol, by priming and loading the chip via the HX IFC Controller (Fluidigm) and performing the QPCR in the BioMark QPCR platform (Fluidigm), with default parameters for gene expression data collection as advised by Fluidigm.

cDNA was generated by subjecting 50 ng of RNA from each sample to reverse transcriptase reaction (Reverse Transcription Master Mix Kit Fluidigm P/N-100-6297). Then, 1.25 µL of each cDNA solution was used to generate a preamp mix containing a pool of the 26 primers pairs and the PreAmp Master Mix Kit (Fluidigm P/N-100-5744). Preamp mixes were run for 14 cycles and the remaining primers were digested with Exonuclease I (New England BIOLAB. P/N M0293l. LOT 0191410). Preamp samples were analyzed for the expression of 22 genes of interest (for primer sequences, see Supplementary Table 3) using the BioMark qPCR platform (Fluidigm, San Francisco, CA, USA). Data were normalized to Gadph, B2m, Actb and Gusb (from the same animal) and fold changes were calculated using the 2-ΔΔCt method⁹⁴ .