2.5. RNA Isolation and qRT-PCR

Total RNA was isolated from the cultured cells (about 5 × 10⁵) using RNAiso Plus Reagent (TaKaRa, Dalian, China) and was reverse transcribed by a ReverTra Ace qPCR RT kit (TOYOBO, Shanghai, China) according to the manufacturer’s protocol. Polymerase chain reaction (PCR) was performed with SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). To release cells from microspheres for RNA extraction, the encapsulating alginate was depolymerized in a 55 mM sodium citrate (Aladdin, Shanghai, China) solution for 30 min [21,25]. To release the cells from the microspheres for RNA extraction, the encapsulated cells were incubated in an appropriate volume of sodium citrate solution (55 mM) at 37 °C for 5 min, then gently pipetted up and down, using a 1 mL pipette tip, a few times until the alginate microspheres were completely depolymerized. The obtained cell suspension was centrifuged at 300× g for 3 min and the supernatant discarded. RNAiso Plus Reagent was then added to the cell pellet for RNA extraction. One μg of total RNA was reverse transcribed into cDNA, using an RT-PCR Kit (FSQ-101, TOYOBO, Shanghai, China), and qPCR was performed using 2× Power SYBR Green Master Mix and an ABI 7500 PCR machine, with GAPDH for normalization of input RNA. RT-qPCR data were analyzed by the 2^−ΔΔCT method. Primer sequences are listed in Supplementary Table S1.