2.2.2. Quantitative RT-PCR

Total RNA samples were extracted from untreated or treated cells with an Absolutely RNA Microprep Kit (Agilent Technologies, Santa Clara, CA, USA). cDNA was generated by using the High-Capacity cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA) and amplified on an Abi Prism 7000 instrument (Applied Biosystems, Foster City, CA, USA) using the Taqman Universal PCR Master Mix (Applied Biosystems). The following human TaqMan gene expression assays were used: HAS2 (Hs00193435\_m1), HAS3 (Hs00193436\_m1), NOS1 (Hs00167223\_m1), NOS2 (Hs01075529\_m1), NOS3 (Hs01574659\_m1), SYND1 (Hs00174579\_m1), SYND2 (Hs00299807\_m1), SYND3 (Hs00206320\_m1), SYND4 (Hs00161617\_m1), NDST1 (Hs00155454\_m1), EXT1 (Hs00609162\_m1), EXT2 (Hs00181158\_m1), and β-actin (Hs99999903\_m1) as the reference gene. The relative quantification of gene expression levels was determined by comparing 2ˆ-∆∆Ct [3,24] using non-treated cells, or the expression of HAS2 and Syndecan-1 as a normalizers.
