Sybr green premix kit

The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay, in accordance with the manufacturer’s instructions, was employed to evaluate the expression levels of apoptosis-related genes in HUVECs. HUVECs were cultured in 12-well plates at a density of 1 × 10⁵ cells per well and incubated for 24 h. Different concentrations of BPNSs (1.25, 5 and 20 μg/mL) were added to the cells and incubated for 24 h. The total RNA was extracted from the cells using the Trizol reagent (Accurate Biology, Changsha, China) and converted to cDNA using the Reverse Transcription Mix Kit (Accurate Biology, Changsha, China). Real-time PCR was performed on a LightCycler 96 real-time PCR system (Roche, Basel, Switzerland) using the SYBR Green Premix Kit (Accurate Biology, Changsha, China). The 2^−ΔΔCT method, with 18S ribosomal RNA (18s rRNA) as the reference gene, was employed to calculate the relative changes in mRNA expression. Three experimental wells were set up for each group. Control groups did not receive BPNS pretreatment. The primers used in this study are listed in Table 1 and were purchased from Sangon Biotech (Shanghai, China).

Total RNA was extracted from sEVs and cells using the miRNeasy Micro Kit (Qiagen, Germany), RNA was reverse transcribed to cDNA using 4 × Reverse Transcription Master Mix (EZBioscience, USA), and qPCR was performed using SYBR Green qPCR Master Mix (EZBioscience, USA). The primers (Sangon Biotech, China) used in this study are listed in Additional file 1: Table S1.
For miRNA analysis, exosomal miRNAs were isolated by using a miRNeasy Micro Kit (Qiagen, Germany), and cDNA for miRNAs was synthesized using miRNA cDNA 1st strand synthesis (Accurate Biotechnology, China). qRT-PCR was performed using a SYBR Green Premix Kit (Accurate Biotechnology, China), which provides miRNA reverse primers. The miRNA-specific forward primers (Sangon Biotech, China) are listed in Additional file 1: Table S2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and small U6 RNA were used as internal references for mRNA and miRNA, respectively.

The reverse transcription–quantitative polymerase chain reaction (RT-qPCR) assay was performed to investigate the adhesion-related gene expression levels of HGFs, which followed the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [27 (link)]. HGFs were seeded at 2 × 10⁵ cells/cm² on the samples in 24-well plates. After 2 and 24 h of incubation, total RNAs were isolated using Trizol reagent (Accurate Biology, Changsha, China), and cDNAs were generated using the Reverse Transcription Mix Kit (Accurate Biology, Changsha, China). Quantitative polymerase chain reaction (qPCR) was conducted using SYBR Green Premix Kit (Accurate Biology, Changsha, China) on a real-time PCR system (LightCycler 96, Roche, Basel, Switzerland). The relative changes in mRNA expression determined from qPCR experiments were calculated by the 2^−ΔΔCT method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. The groups without UVC pretreatment were set as the controls. The primers (Tsingke Biotechnology, Beijing, China) used are shown in Table 1.