7500 fast sds program v 2

Total RNA was extracted from cultured primary AF/NP cells using the PureLink™ RNA Mini Kit. A total of 50 ng of RNA was reverse-transcribed using a high capacity cDNA RT kit to obtain cDNA (1). To determine gene expression profiles, all genes were amplified using TaqMan Gene Expression Assays for CHAD, β-actin (ACTβ), COMP, MMP-7, MMP-19, and IL-1β. qPCR analysis was performed on an Applied Biosystems 7300/7500 real-time PCR system (Thermo Fisher Scientific, USA) with a reaction mixture consisting of 1 μl TaqMan Gene Expression Master Mix, 10 μl TaqMan Gene Expression Master Mix, 4 μl cDNA template, and UltraPure Dnase/Rnase free distilled water for each gene in MicroAmp Fast Optical 96-Well Reaction Plates under the following thermocycling conditions: 2 min at 50˚C, 10 min at 95˚C, 15 sec at 95˚C and 1 min at 60˚C for 40 cycles for each duration (1) . As a result of the qPCR experiment, the relative quantity values of each sample were obtained using the 7500 Fast SDS program V.2.3 (Thermo Fisher Scientific, USA). ACTβ was used as an endogenous control to normalize the targeted gene expressions (1) . For obtaining comparative results, a reference sample (Group 1, 0 h) was used, and relative quantity values were calculated using the 2 -∆∆Cq method.

Total RNA was extracted from cultured primary human chondrocytes using the PureLink RNA mini kit. The quantity of RNA obtained from each sample was measured using a UV spectrophotometer. The cDNA was obtained by reverse transcription of 50 ng RNA using a high capacity cDNA reverse transcription kit and a thermal cycler. All genes were amplified using TaqMan ® Gene Expression assays for CHAD, ACTβ, COMP, MMP-7, MMP-19, and IL-1β.The qPCR was performed on an Applied Biosystems 7300/7500 real-time PCR system (Thermo Fisher Scientific, Inc.), and the reaction mixture consisted of 1 μlTaqMan gene expression assay, 10 μlTaqMan gene expression master mix, 4 μl cDNA template, and UltraPure DNase/RNase free distilled water for each gene, conducted in MicroAmp fast optical 96 well reaction plates. The thermocycling conditions were as follows: 2 min at 50˚C, 10 min at 95˚C, 15 sec at 95˚C, and 1 min at 60˚C, for 40 cycles. The RQ values of each sample from the RTqPCR experiment were obtained using the 7500 FastSDS program V.2.3 (Thermo Fisher Scientific, Inc.
). An endogenous control (ACTβ) was utilized to normalize the target gene expression (9) (10) (11) (12) (13) 21) .