Validating Gene and miRNA Expression

To validate gene expression changes, 1 μg of total RNA was used for cDNA synthesis with RevertAid RT Kit (ThermoFisher Scientific, USA) according to manufacturer’s instructions. To evaluate miRNA expression, 0.2 μg of total RNA was used to perform cDNA synthesis with RevertAid RT Kit (ThermoFisher Scientific, USA) as described previously [16 (link)]. Quantitative real-time PCR (qRT-PCR) was performed on Eco™ RT-PCR system (Illumina, Inc.) using 2x Maxima SYBR Green qPCR MasterMix (ThermoFisher Scientific, USA) according to manufacturer’s instructions. The relative changes in gene and miRNA expression were calculated by ∆∆C_t method comparing expression levels in LLC1 cells grown under 2D and lr-ECM 3D or LLC1 tumors with hprt1 or sno135 as endogenous controls for expression normalization, respectively. The primer sequences used for microarray data validation are shown in Additional file 1: Table S1.