*2.7. RNA Extraction and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)*

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<sup>t</sup> method was employed to determine the relative fold change in gene expression levels between heparanase-treated and untreated cells.
