2.8. Quantitative Polymerase Chain Reaction (qPCR)

RNA extraction was done using RNeasy Microkit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. The reverse transcription to the complementary DNA (cDNA) of 1 μg of RNA was completed using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (ThermoScientific).

The qPCR was performed with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) with custom primers ordered from Integrated DNA Technologies (IDT, Newark, NJ, USA). The fold change in gene expression was calculated using the −∆∆C_t method with reference to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene and compared to the control growth medium groups in the monolayer, static, and dynamic cultures. Gene primer sequences are listed in Table 1.

Gene primers and sequences for RT-PCR.