Sybr green quantitect primer assay

RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA, USA) and cDNA was generated from 1 ug of RNA using SuperScript III First-Strand Synthesis System (Invitrogen, Grand Island, NY, USA). Quantitative PCR was performed on 8 samples using SYBR Green QuantiTect Primer Assay (Qiagen) according to the manufacturer’s instructions in a 7900HT Fast-Real Time PCR System Instrument (Applied Biosystems, Grand Island, NY, USA). Primer pairs for the individual genes were obtained from the bioinformatically validated QuantiTect Library and are as follows: PRKCI (QT00058954) and CPT1A (QT00082236). The fold changes in gene expression were calculated using the delta–delta CT method (assays were performed in triplicate).

To determine the effects of the heparanase treatment on gene expression, RNA was extracted using the RNeasy^® mini kit (Qiagen, Manchester, UK) and cDNA was generated using a High-Capacity cDNA Reverse Transcriptase Kit^® (Applied Biosystems, Loughborough, UK) according to the manufacturers’ protocols. RT-qPCR was performed on a QuantStudio 3 real-time PCR system (Applied Biosystems) using SYBR green QuantiTect primer assays (Qiagen) to assess the gene expression of Sox-9 (SOX9), aggrecan (ACAN), collagen type II (COL2A1), fibroblast growth factor receptor 3 (FGFR3), collagen type X (COL10) and activin receptor-like kinase (ALK-1). Peptidylprolyl Isomerase A (PPIA) and TATA-box binding protein (TBP) were used as reference genes and the delta-delta C_t method was employed to determine the relative fold change in gene expression levels between heparanase-treated and untreated cells.

Total RNA of mucosal tissue samples were extracted by the use of Trizol (Life Technologies, Germany), according to the manufacturer’s instructions. cDNA was synthesized by the use of the High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Germany). Expression of mRNA was quantified in triplicates by a real-time (RT)- PCR by SYBR Green QuantiTect Primer Assays (IL-1α, QT00113505; IL-1R1, QT00095256; Qiagen) or TaqMan probes (housekeeping gene GAPDH, NM_008084.2; IL-1β, Mm00434228; Thermo Fisher Scientific, Germany). Primers were designed for AMPs listed in Table 1. Quantitative PCR was performed with SYBR Green PCR Master Mix or TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, Germany).

The expression of chondrogenic genes Sex-Determining Region Y-Box 9 Protein (SOX9), aggrecan (ACAN), collagen type II (COL2A1) and frizzled-related protein 1 (FRZB) and hypertrophy genes collagen type X (COL10A1) and activin receptor-like kinase 1 (ALK1) [19 ], were assessed by qRT-PCR at passage 3. Briefly, messenger RNA was isolated from cultured cells using an RNeasy kit (Qiagen) and cDNA was generated using the High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems), according to manufacturer's recommendations. qRT-PCR analysis was conducted on a Quant Studio 3 Quantitative Real-Time PCR System (Applied Biosystems) using SYBR green QuantiTect primer assays for SOX9, ACAN, COL2A1, FRZB, COL10A1, ALK1 (Qiagen). Relative gene expression was determined by the comparative C_t method (2 ^−ΔCt), using the reference genes peptidylprolyl Isomerase A (PPIA) and TATA-box binding protein (TBP). These reference genes were the two most stable from a screening test of five genes (data not shown).