Taqman probes specific for the

For quantification of mtDNA, total DNA was isolated from heart tissue using the DNeasy Blood & Tissue Kit (QIAGEN). Semiquantitative RT-PCR was carried out on 4 ng of total DNA in a 7900HT Real Time PCR system (Applied Biosystems), using TaqMan probes specific for the CoxI and 18S genes (Applied Biosystems).

Real-time quantitative PCR (qRT-PCR) was performed using TaqMan probes specific for the target genes (Applied Biosystems, Waltham, USA): TNF (Hs00174128_m1), TNFR1 (Hs01042313_m1), TNFR2 (Hs00961750_m1), ADAM17 (Hs01041915_m1), ADAM10 (Hs00153853_m1), TGF-β (Hs00248373_m1), and TIM3 (Hs00958618_m1). 18S (18S ribosomal RNA gene) (Hs03928990_g1) and ACTB (β-actin) (Hs01060665_g1) were used as endogenous controls. Single reactions were prepared with the Maxima Probe/ROX qPCR Master Mix (No. Cat. K0231, Thermo Fisher Scientific, Waltham, USA), cDNA was diluted 1:4 (9 ng/µL to 2.25 ng/µL), and all amplifications were run by duplicate, under the following thermal conditions: 95 °C for 10 min followed by 40 cycles of 60 °C for 1 min and 95 °C for 15 s, with the StepOnePlus^TM Real-Time PCR Systems (Applied Biosystems, Waltham, MA, USA). The relative quantification of transcripts was quantified using the ΔΔCT method. The results were reported as the n-fold change for each target gene in each experimental group, normalized with the endogenous controls ACTB and 18S, and relative to a control group of ten HD (2^−ΔΔCT = 1).