Abi qpcr model 7300

mRNA was isolated using RNeasy mRNA purification columns (Qiagen, Venlo, Netherlands). cDNA was prepared using the OneStep RT–PCR kit (Invitrogen, Waltham, Massachusetts, USA). qPCRs were performed using an ABI qPCR model 7300 or 7900 (Applied Biosystems, Waltham, Massachusetts, USA) with purified samples containing a SYBER Green mix (Applied Biosystems). Primers were as follows: SIRT1‐forward: CAGATTAGTAGGCGGCTTGA; reverse: CTAA ACTTGGACTCTGGCAT; COL2A1 (Exon 2)‐forward: GAGCCCTGCCGGATC TGT; reverse: GAGGCAGTC TTTCACGTCTTC; Matrix Metallopeptidase 13 (MMP13)‐forward: AGTTTGCAGAGCGCTACCTGAGAT; reverse: TTTGCCAGT CACCTCTAAGCCGAA; ADAMTS5‐forward: TTCAACGTCAAGCCATGGCAA CTG; reverse: TGACGATAGGCAAACTGCACTCCT; SOX9‐forward:AGGCAA GCAAAGGAGATGAA; reverse: TGGTGTTCTGAGAGGCACAG; ACAN‐forward: TGCGGGTCAACAGTGCCTATC; reverse: CACGATGCCTTTCACCAC GAC. Values were normalized to those obtained for human beta 2‐microglobulin (B2MR) or glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), which were unaffected by the experimental treatments: B2MR‐forward: ACCCCCACTGAAAAAGATG AG; reverse: ATCTTCAAACCTCCATGATGC; GAPDH‐forward: TACTAGCGGT TTTACGGGCG; reverse: TCGAACAGGAGCAGAGAGCGA. Mouse Gapdh: forward: TGCCCCCATGTTTGTGATG; reverse: TGTGGTCATGAGCCCTTCC. Mouse Acan‐forward: GCTGGCTGACCAGACAGTCA; reverse: CCGGATTC CGTAGGTTCTCA.

mRNA was isolated using RNeasy mRNA purification columns (Qiagen, Hilden, Germany). cDNA was then prepared using the OneStep RT-PCR kit according to the manufacturers guidelines (Invitrogen Carlsbad, CA, USA). Real-time PCR reactions were performed using an ABI qPCR model 7300 or 7900 (Applied Biosystems, Foster City, CA, USA) with purified samples containing a Syber Green mix (Applied Biosystems) in accordance to the manufacturers’ guidelines. Primers for quantitative PCR were prepared for the following human genes: Cathepsin S(CAT), forward: 5′-GACTGGAGAGAGAAAGGGTGTGTT-3′, reverse: 5′-CAGCTTTCCTGTTTTCAGCTTCA-3′, hβ2MG-F: ACCCCCACTGAAAAAGATGAG; reverse: ATCTTCAAACCTCCATGATGC; GAPDH-F: TACTAGCGGTTTTACGGGCG; R: TCGAACAGGAGCAGAGAGCGA.