Heart tissue sections (10 mg) were subjected to Proteinase-K lysis and total DNA was extracted by phenol/chloroform extraction/ethanol precipitation method. Total DNA (100 ng) was treated with RNase A (EN0531, Thermo Scientific, Waltham MA), purified by using DNeasy Mini Spin Columns (Qiagen, Germantown MD), and examined for quality (OD260/OD280 ratio of 1.7–2.0) and quantity ([OD260 –OD320] x 50-?g/ml) by using a DU 800 UV/visible spectrophotometer. To assess the cardiac mtDNA integrity, total DNA was used as template with mtDNA-specific primer sets and PfuUltra II Fusion HS DNA polymerase (Stratagene, La Jolla CA). The long range PCR to amplify 10 kb mtDNA was performed with hot start of 2 [email protected]°C followed by [email protected]°C, [email protected]°C, [email protected]°C for 28 cycles. Densitometry analysis of mtDNA bands was performed as above, and 10 kb mtDNA level was normalized to short-length, 117-bp mtDNA and 96-bp GAPDH nuDNA.
To assess tissue parasite burden, total DNA (100 ng) was used as a template with SYBR Green Supermix (1708882, Bio-Rad) and Tc18SrDNA-specific primers, and real-time qPCR was performed on an iCycler thermal cycler. Data were normalized to GAPDH, and relative parasite burden was calculated as 2-?Ct (?Ct = CtTc18SrDNA—CtGAPDH). The primers are listed in Table B of S1 Table.