RNA Isolation and qRT-PCR Analysis

Hydrogels’ treated cells lysate was used for RNA isolation by using a commercially available kit (Gene JET RNA Purification) according to the manufacturer protocol (Thermo Scientific, # K0731, USA). RNA was extracted in triplicates [34 (link)]. For reverse transcription, cDNA was synthesized by using a commercially available kit (RevertAid™ H Minus First Strand) by following the manufacturer’s instructions (Thermo Scientific, # K1622, USA). Amplification was done by real-time PCR (Applied Biosystems 7900HT Fast Real-Time PCR System) using kit (Maxima SYBR Green/ROX qPCR Master Mix (2X), Thermo Scientific, USA), following manufacturer’s guidelines (Thermo Scientific, # K0221, USA). The relative expression levels of each target gene were calculated using the Ct method by using Beta-actin (β-actin), and Relative mRNA expressions were analyzed using the ΔΔCt equation.