Sybergreen mix

To analyze the gene expression of the enzymes, Real Time quantitative PCR (RT-qPCR) and conventional RT-PCR were used. Primers were designed using the open-source Perlprimer program [51 (link)] and the NCBI Primer-BLAST tools. The primers for MMP-2, MMP-9, and MMP-14 were purchased from Invitrogen, Thermo Fisher Scientific, MA, USA and were initially tested using conventional RT-PCR and then confirmed by RT-qPCR. The real-time reactions were prepared containing Syber Green Mix (Applied Biosystems, Thermo Fisher Scientific, Walsham, MA, USA). The amplifications were performed using the 7300 REAL TIME PCR system (Applied Biosystems, Thermo Fisher Scientific, Walsham, MA, USA). Dissociation curves verifying amplification specificity were also performed. To evaluate the differential expression of the treated groups, the relative quantification method with 18s was used an endogenous control. The details of this protocol have already been established in a previous publication [34 (link),37 (link)]. Primer sequences used were as follows:-
MMP2 forward, GAC CAG AAT ACC ATC GAG ACC A; MMP2 reverse, GTG TAG CCA ATG ATC CTG TAT GTG; 128 bp
MMP 9 forward, TTT GTT CAA GGA TGG GAA GTA CTG; MMP 9 reverse, CTC CTC AAA GAC CGA GTC CA; 124 bp
MMP 14 forward, CTT CAA AGG AGA CAA GCA TTG G; MMP 14 reverse, CCC TTG TAG AAG TAA GTG AAG AC; 297 bp

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.