Real time thermal cycler

The upper PDMS membrane was removed gently with tweezers, and the cells were digested to extract RNA for reverse transcription sequencing after continuous perfusion culture for 48 h. Total RNA was extracted from 0 to 1000 ng/mL PLP-stimulated A-MSCs using TRIzol reagent (Invitrogen, 15596018, Carlsbad, CA, USA) and subsequently reverse transcribed into cDNA. qRT-PCR analysis was performed to measure mRNA expression of IDO1, TLR3, Toll-like receptor 4 (TLR4), Toll-like receptor 6 (TLR6), and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). qRT-PCR was performed using the Real-Time Thermal Cycler (Analytik Jena AG, qTOWER3G, Germany), and 0 ng/mL PLP-treated cells served as the control group. Reverse transcription was performed using PCR Kits from TOYOBO (Code Nos. FSQ-101 and QPK-201), according to the manufacturer's protocol. The primer sequences used for quantitative RT-PCR are listed in Table 2.

Three replicates of qRT-PCR were performed for each sample to verify the DEGs detected by the transcriptome sequencing. We selected six genes for validation and analysis. We used Primer Premier 5.0 software (http://www.premierbiosoft.com/primerdesign/) to design primers (Table A1). Primers were synthesized in the Shanghai Shenggong Company. We synthesized cDNA according to the manufacturer's instructions using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). QRT-PCR was performed in 15 μl reactions on the Real-Time Thermal Cycler (Analytik Jena AG, qTOWER³G, Germany). The template was 1.5 μl cDNA of the first strand. The rest were 7.5 μl of 2 × UltraSYBR Mixture (with ROX) (CWBIO, Beijing, China), 1.2 μl 10 μM forward primer, 1.2 μl 10 μM reverse primer, and 3.6 μl of ddH₂O. The reference gene was GhUB7. The qRT-PCR program was set as follows: preincubation at 95°C, 10 min first; the second step was amplification at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s, 38 cycles in total. A melting curve analysis was performed in order to evaluate the primer's specificity. The melting curve analysis program was set as follows: 95°C for 15 s, 60°C for 60 s, 95°C for 15 s, and 60°C for 15 s.