NSCLC miRNA and mRNA Expression Analysis

For all cell lines, RNA extraction and RT-qPCR experiments were conducted as previously described.^{22 (link)} From each formalin-fixed, paraffin embedded patient sample, RNA was extracted from a 1 × 7 μm section using the miRNeasy FFPE kit (217504, Qiagen, Germantown, MD, USA). miR expression analysis was performed using the miRCURY LNA Universal RT microRNA PCR system (203301, Exiqon, Woburn, MA, USA), whereas mRNA expression analysis was performed using the High Capacity Reverse Transcriptase Kit (4368813, Life Technologies) and TaqMan PreAmp Master Mix kit (4384267, ThermoFisher) according to manufacturer’s protocol. All RT-qPCR was performed in technical cDNA and qPCR duplicates using either hsa-miR-103a-3p and hsa-miR-423-5p or IPO8 and PUM1 as reference genes, as they have previously been reported stably expressed in NSCLC.^{25 (link), 26 (link)} All data was analyzed using NormFinder to ensure stability of the reference genes.^{27 (link)} For each sample, relative quantities were calculated as 2^−ΔCt and determined as the average relative quantities in the cDNA synthesis duplicates.