qPCR validation of StringDB PPI hub gene and IPA prediction outcomes

qPCR was performed with CFX96 Real-Time PCR Machine (Bio-Rad) to validate NanoString gene expression levels of the key hub genes identified from StringDB PPI analysis. Gene expression for important molecules from IPA upstream regulator and biofunctional or disease prediction outcomes were also validated via qPCR. Mouse-specific primer sequences were designed based on NanoString target probe sequences using ApE, a plasmid editor (Table ​(Table11)¹⁰⁷. qPCR was carried out using iTaq™ Universal SYBR® Green Supermix (Catalog no.1725120; Bio-Rad, Hercules, CA, USA) in addition to template cDNA and specific primers. All qPCR experiments were conducted in duplicates with a sample size of n = 3 per group. Relative quantitative analysis of gene expression was completed using the standard comparative Ct method (2^−ΔΔCt) by using the recorded raw Ct values of the gene of interest and housekeeping gene¹⁰⁸. Using RefFinder, Gusb was selected as the most stable reference gene for qPCR normalization based on expression stability assessment among the other common candidate reference genes, Gapdh, Hprt, and Tubb5, present in the NanoString panel^109,110.

Primers sequences for qPCR.