Sybr qpcr master mix

To ensure the credibility of the RNA-seq sequencing data, a quantitative reverse transcription-PCR (RT-qPCR) analysis was performed to authenticate the DEGs detected from transcriptome sequencing. For validation purposes, ten DEGs were randomly chosen from the RNA-seq sequencing data: NOS2, TNFAIP3, CTSL, MMP9, CSF3, IL1R2, CCL20, TNFRSF13B, F3, and CXCR2. These DEGs played key roles in various signaling pathways, including antigen processing and presentation, interaction between cytokines and cytokine receptors, the calcium signaling pathway, PI3K-Akt signaling pathway, complement and coagulation cascades, NF-κB signaling pathway, the TNF signaling pathway, the Toll-like receptor signaling pathway, and the IL-17 signaling pathway.—PBMC samples infected with BVDV were prepared according to the previously mentioned method, while control samples consisted of PBMC samples without any infection. The total RNA was extracted and subjected to SYBR Green-based qRT-PCR using Roche SYBR qPCR Master Mix (Basel, Switzerland) and the primers listed in Table 1. The qRT-PCR analysis was carried out using the ABI 7500 system (Applied Biosystems, Foster City, CA, USA), with the ACTB gene serving as an internal reference gene. The 2^−ΔΔCt method was applied to determine the relative expression levels of the target genes. Each qRT-PCR assay was performed in four technical replicates.

Total RNA was extracted from the cecum and adhesion tissue of rats, using TRIzol reagent (Servicebio, China). RNA concentration and purity were measured using Nanodrop 2000, and the overconcentrated RNA was diluted in an appropriate ratio to a final concentration of 200 ng/μL. cDNA was synthesized from 2 μg of total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo, USA). For real-time PCR, the 0.2 mL PCR tube was prepared to prepare the following reaction system, and all reactions were performed in triplicate: 2× SYBR qPCR Master Mix (Roche, Switzerland) (12.5 μL), 7.5 μM gene primer (2.0 μL), reverse primer (2.5 μL), and ddH2O (8.0 μL). The reaction conditions were as follows: the predenaturation was 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s performed with ABI StepOnePlus (Applied Biosystems). All results were processed by the double-delta method (2^−ΔΔCt). The primers (provided by Servicebio) are shown in Table 3.

The hDPCs seeded at 10⁵ cells per well in 6-well plates were cultured in the extract of TDM, PAA-CMC-nHA, PAA-CMC-TDM, and CMC for 7 and 14 days. The total ribonucleic acid of the induced cells was extracted with the Cell/tissue Total RNA Isolation Kit and reverse-transcribed into cDNAs through the cDNA Synthesis Kit. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using the LightCycler PCR System (Roche) with SYBR qPCR Master Mix. The cycle threshold (Ct) was recorded after each reaction and used in the 2^–ΔΔ Ct method to calculate the relevant date with GAPDH as the loading control gene. The primer sequences are listed in Supplementary Table 1. All experiments were performed in triplicates.

Total RNA from cells was prepared using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Mouse back skin tissues were separated to dermis and epidermis by incubating in dispase solution (5 mg/ml in DMEM with 10% FBS) at 37 °C for 2 h, and homogenized in Trizol reagent (Invitrogen) using a homogenizer (Ultra-Turrax Dispenser, IKA) and total RNA was extracted according to manufacturer’s instructions. 1 µg of total RNA was reverse transcribed to cDNA with oligo dT and Superscript II (Invitrogen). Real-time PCR was performed using CFX96 real-time system (Bio-Rad) and SYBR qPCR master mix (Kapa biosystems). Real-time PCR primer sequences are listed in Supplementary Table S2.