RNA Extraction and qPCR Analysis of Hydrogel Constructs

Hydrogel constructs were snap frozen on dry ice / isopropanol and stored at −80 °C before RNA extraction following our reported methods.^{32 (link)} Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) and reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time quantitative polymerase chain reaction (qPCR) was carried out using an ABI 7300 real-time PCR system where cDNA templates were combined with primers (400 nM) and Power SYBR green master mix. The relative expression levels of selected genes were monitored with primers listed in SI Table 1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference. The fold changes were calculated with the Pfaffl method, using qbase+ software (Biogazelle, Zwijnaarde, Belgium). A total of three biological repeats were analyzed for each gel composition.