Rna purification kit

RNA was isolated by RNA Purification Kit (Cat#RN001, EZBioscience) and reverse transcribed by HiScript III RT SuperMix reverse-transcription reagent kit (Cat#711-02/03, Vazyme Biotech Co.). qPCR was performed on a LightCycler 96 Real-Time PCR System (F. Hoffmann-La Roche) using FastStart Essential DNAGreen Master assay (F. Hoffmann-LaRoche). The primers used are listed in Supplementary Table 1. All of the primers were synthesized by Sangon, Shanghai.

Total RNA was extracted from cells using an RNA Purification Kit (EZ Bioscience) in accordance with the manufacturer's protocol, followed by measurement of RNA concentration. For reverse transcription mRNAs of ALP, Runx2, CCAAT/enhancer binding protein α (C/EBPα), CD31 and VEGF, cDNA was synthesized from 1 μg of total RNA using a color reverse transcription kit (EZ Bioscience). Then quantitative PCR was performed using a SYBR Green PCR master mix kit (EZ Bioscience). GAPDH was used as the reference gene, and the primer sequences (BioTNT) of the genes above are listed in Table S1. The following cycling conditions were utilized for RT-PCR: 95℃ for 5 min, followed by 40 cycles at 95℃ for 10 s and 60℃ for 30 s. The relative expression of mRNAs was calculated by the 2^-△△Ct method.

Total RNAs were extracted using an RNA Purification Kit (EZBioscience, B0004). After removing genomic DNAs with a DNA remover, total RNAs were reverse transcribed into cDNAs using the Reverse Transcription Kit (EZBioscience, A0010GQ). cDNA amplification was performed using the TB Green® Premix Ex Taq™ II (TaKaRa, RR820A). The 2^−ΔΔCt method was used to calculate gene expression levels relative to GAPDH. Primers were listed in Table S1.

Gene expression related to angiogenic growth factors was evaluated by qRT-PCR. hADSCs (1 × 10⁵) were seeded into PADM, FADM, LADM and FLADM of a well size of a 24-well plate and cultured for 48 h. Total RNA was extracted and reverse transcribed into cDNA with PrimeScript RT Master Mix using RNA purification kit and Reverse Transcription kit (with DNase) (EZBioscience). Gene expression levels of vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF) [24 (link)] were normalized to that of GAPDH and quantified with the comparative Ct method. The primers synthesized by Sangon Biotech Co. (Shanghai, China) are mentioned in Table 2.

Equal amounts of protein lysates (20 µg) extracted from cells transfected with NC and small interfering (si) IL4I1 were subjected to SDS-PAGE and transferred to PVDF membranes. Following incubation with 5% skimmed milk for 1 h at room temperature (RT), PVDF membranes were exposed to primary antibodies targeting IL4I1 (1:1,000, ab222102, Abcam), CD11B (1:1,000, ab133357, Abcam), CD204 (1:1,000, ab271070, Abcam), CD206 (1:2000, 60143-1-Ig, Proteintech), CD163 (1:1,000, ab182422, Abcam), CD86 (1:1,000, ab239075, Abcam), and beta-tubulin (1:2000, DF7967, Affinity) overnight. Subsequently, PVDF membranes were incubated with Goat Anti-Rabbit/Mouse IgG (H+L) HRP (1:5,000, Affinity) for 1 h. Chemical imaging was developed using ECL solutions (Epizyme, Shanghai, China). Pictures were obtained using a Bio-Rad imaging system.
The extraction of total RNA from cells transfected with NC and siIL4I1 was done through an RNA Purification Kit (B0004D, EZBioscience, USA). Subsequently, reverse transcription was carried out using RNA (1 μg). After ward, RT-qPCR was done using the SYBR qPCR Master Mix kit (Epizyme, Shanghai). Amplification was detected using QuantStudio (Thermo Scientific, USA). The primer sequences we used are listed in Table 3.