Quantitative RT-PCR was performed to quantify the expression of ENS mucosal and substance P (SP) cascade markers in mono-culture and co-culture silk scaffolds. The mRNA was isolated from samples using the aforementioned method. The mRNA samples were then reverse transcribed to cDNA using the High Capacity Reverse Transcription kit (ThermoFisher, Waltham, MA) in 20 μL reactions containing 400 ng of mRNA, according to the manufacturer’s instructions. Amplification was carried out as follows: 10 minutes at 25°C, 120 minutes at 37°C, and then 5 minutes at 85°C.
Quantitative RT-PCR was performed using Taqman fluorescent hybridization probes (ThermoFisher, Waltham, MA) according to the manufacturer’s instructions. The reactions were incubated at 95 °C for 10 min followed by 2 min at 50°C. Amplification was then carried out with 50 cycles of 15 seconds at 95°C and 1 min at 60 °C. Samples with Ct values higher than 40 cycles were counted as no expression. Relative mRNA expression was then calculated using a comparative cycle threshold method (ΔΔCt). All genes and experimental groups were normalized using 18S as a housekeeping gene. Mucosal markers used include Muc-2 and Muc5AC, and substance P cascade markers include TAC1 and NK-1.
Quantitative RT-PCR was performed using Taqman fluorescent hybridization probes (ThermoFisher, Waltham, MA) according to the manufacturer’s instructions. The reactions were incubated at 95 °C for 10 min followed by 2 min at 50°C. Amplification was then carried out with 50 cycles of 15 seconds at 95°C and 1 min at 60 °C. Samples with Ct values higher than 40 cycles were counted as no expression. Relative mRNA expression was then calculated using a comparative cycle threshold method (ΔΔCt). All genes and experimental groups were normalized using 18S as a housekeeping gene. Mucosal markers used include Muc-2 and Muc5AC, and substance P cascade markers include TAC1 and NK-1.