Validating Gene Expression in Tissue Biopsies

The expression of the genes identified by in silico analysis was validated by RTq-PCR for cDNA obtained from the archival tissue biopsies, plasma, and cells. Approximately 50 ng of the gene-specific cDNA obtained from AS, LC, and AC tissues, as well as lung cancer and asthmatic cells and plasma, was added to 2× maxima SYBR green master mix (Thermo Fisher Scientific, Waltham, MA, USA) along with the primers as listed in Supplementary Table S1. The reaction was carried out in a Quant Studio 3 cycler (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The cycling conditions were an initial single hold stage at 50 °C for 2 min, 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s; there was then a melt-curve stage: 60 °C for 1 min and 95 °C for 1 s. Each cDNA reaction was performed in triplicate, and each experiment was repeated three times along with a negative cDNA sample and a negative non-template control for each pair of primers. The Ct value of the gene of interest was normalized against the expression of the housekeeping gene (18S) for each sample, and the relative gene expression (2^−ΔΔCt) was derived from the ΔCt values [32 (link),33 (link)]. The fold-expression values were normalized to log₂, and the relative expression of each gene was compared between the groups.