Sybr green system

The myofibroblast transformation of keratocytes was explored by real-time polymerase chain reaction (RT-PCR). In brief, keratocytes were seeded onto collagen films and cultured for 3 days. Then 2.5 ng ml⁻¹ recombinant human TGF-β1 was added and cells were cultured for another 3 days. Then RNA was extracted using the HiPure Total RNA Micro Kit following the manufacturer's instructions. Total RNA concentrations were quantified using NanoDrop 2000 (Thermo Scientific, USA). Next, a reverse transcription reagents kit was used to synthesize cDNA. Finally, RT-PCR was achieved using the SYBR green system (Genecopoeia, USA). Amplifications for cDNA samples were carried out at 50 °C for 2 min and at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The following primer sequences were used: GAPDH: forward: 5′-ACTTCGGCATTGTGGAGG-3′; reverse: 5′-GGAGGCAGGGATGATGTTCT-3′; for α-SMA: forward: 5′-CCATGCCATCATGCGTCT-3′; reverse: 5′-GCCATCTCGTTTTCAAAGTCC-3′; for Col1A1: forward: 5′-GCCTGAGCCAGCAGATTGA-3′; reverse: 5′-AGGTTGCCCCAGTGTCCAT-3′; CTGF: forward: 5′-AGGAGTGGGTGTGTGATGAG-3′; reverse: 5′-CCAAATGTGTCTTCCAGTCG-3′. The relative quantification of target genes was normalized to the corresponding value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculated using the 2^−ΔC_t method.

The marker genes of osteogenic differentiation (Runx 2, COL I, ALP, OPN and OCN) in the hFOB cells were detected by quantitative real-time PCR. The hFOB cells were seeded on the scaffolds at a density of 2 × 10⁵ cm⁻² and subsequently cultured for 3, 7 and 14 days at 37 °C. The total RNA was isolated using a HiPure Total RNA kit (Magentec, China) at the specified time intervals. The RNA was then reverse-transcribed into cDNA using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa Biotechnology, Japan). Finally, qRT-PCR analysis was performed using a SYBR Green system (GeneCopoeia) on a RT-PCR instrument (QuantStudio 6 Flex, Life Technologies). The relative quantification of the target genes was normalized to that of beta actin, and the 2^−ΔΔC_t method was used to calculate the gene expression.

Total RNA was extracted from cells with TRIzol reagent (Invitrogen, USA) and quantified with a spectrophotometer (Thermo Scientific, USA). Subsequently, the PrimeScript RT reagent kit (TaKaRa Biotechnology, Japan) was used to perform reverse transcription. Finally, qPCR was conducted on a QuantStudio 6 Flex system (Life Technologies, USA) using the SYBR Green system (Gene Copoeia, USA) to quantify the expression of relative genes. The relative quantification of target genes was normalized to that of GAPDH, and the 2^−ΔΔCt method was used to calculate the fold changes. The primers used in the present study were synthesized by TaKaRa and their sequences are listed in Tables S1–S3.