Quantifying Enzyme Gene Expression

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