Quantitative real-time PCR

Total RNA was isolated from cells using the InnuPREP RNA mini kit (Analytikjena, cat. no. 845-KS-2040250, Jena, Germany). It was transcripted into cDNA carried out with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat. no. 4368814, Foster City, CA, USA) following the supplier´s protocols. qPCR was performed in a 7300 real-time PCR detection system (Applied Biosystems) with RT2 SYBR Green qPCR Primer Assay (Qiagen, cat. no. 330500, Hilden, Germany) and Takyon™ ROX probe qPCR Kit (Eurogentec GmbH, cat. no. UF-RPMT-B0100, Cologne, Germany). HAS2 knockdown was confirmed using the TaqMan probe HS00193435 m1 (Applied Biosystems). Results were evaluated using the 2^−∆∆Ct method. β-actin samples were used as internal controls. The fold change shows the expression of the investigated enzymes in HAS2 knockdown cells compared to the control samples. Primer sequences are shown in Supplementary Table S1. The results were formed out of 7 experiments with double or triple replicates in each experiment.