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.
According to the World Health Organization (WHO), there are approximately 1.2 billion adolescents in the world between the ages of 10 and 19. The WHO documented the statistics regarding teen death, stating that “road injuries were the leading cause of death in 2015, followed by lower respiratory infections and suicide.”.
His twin shot dead, a Washington teen shines spotlight on gun crime.