Taq green master mix

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). An RNA denaturation mix consisting of isolated RNA, oligo dT primers, and nuclease-free water was denatured. Reverse transcription polymerase chain reaction (RT-PCR) was performed using oligonucleotide primers specific to MMP-1 (forward primer, TGC AAC TCT GAC GTT GAT CCC AGA; reverse primer, ACT GCA CAT GTG TTC TTG AGC TGC; 122 bp), to MMP-3 (forward primer, CTG GGC CAG GGA TTA ATG GAG; reverse primer, CAA TTT CAT GAG CAG CAA CGA GA; 102 bp) or to MMP-9 (forward primer, ATT TCT GCC AGG ACC GCT TCT ACT; reverse primer, CAG TTT GTA TCC GGC AAA CTG GCT; 195 bp). Two reactions were run in parallel with a second reaction containing only Platinum Taq polymerase to assure that the source of the RT-PCR product was mRNA. Complementary DNA was synthesized by adding prime RT premix, Taq Green Master Mix (Thermo Scientific, Carlsbad, CA, USA), and 10 pM of each forward and reverse primer. The amplification reaction was carried out for 30 cycles on a DNA Engine Cycler (Bio-Rad, Hercules, CA, USA). The amplified PCR products were analyzed using Multi Gauge software (Fujifilm, Tokyo, Japan) after electrophoresis. The level of b-actin was used as an internal standard.

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). An RNA denaturation mix consisting of isolated RNA, oligo dT primers, and nuclease-free water was denatured. Reverse transcription polymerase chain reaction (RT-PCR) was performed using oligonucleotide primers specific to eNOS (forward primer, 5′-CTG GCT TTC CCT TCC AGT TC-3′, 225 bp, reverse primer 5′-CCT TCC AGA TTA AGG CGG AC-3′, 225 bp) or MMP-2 (forward primer, 5′-CTC GTG CCT TCCT AAG TCT GG-3′, 251 bp, reverse primer, 5′-GGC GTT CCC ATA CTT CAC AC-3′, 251 bp). Two reactions were run in parallel with a second reaction containing only Platinum Taq polymerase to assure that the source of the RT-PCR product was mRNA. cDNA was synthesized by adding prime RT premix, Taq Green Master Mix (Thermo Scientific, Carlsbad, CA, USA), and 10 pM of each forward and reverse primer. The amplification reaction was carried out for 30 cycles on a DNA Engine Cycler (Bio-Rad, Hercules, CA, USA). The amplified PCR products were analyzed using Multi-gauge software (Fujifilm, Tokyo, Japan) after electrophoresis. The level of b-actin was used as an internal standard.