96cfx real time system

Absolute telomere length was determined according to published protocol (O'Callaghan & Fenech, 2011). DNA from spleen and liver was isolated using DNeasy Blood and Tissue kit (Qiagen) with slight modification of adding 1 mm DTT to eluate. Briefly, a standard curve of known quantities of a synthesized 84 mer oligonucleotide containing only TTAGGG repeats was generated based on CT (cycle threshold) using iQ SYBR Green Supermix (Bio‐Rad). A standard curve using the single copy gene, 36B4, was generated to serve as a control for amplification and to determine genome copies per sample. For both standard curves using synthesized oligomers, 20 ng of plasmid DNA (pBR322 Vector, New England Biolabs) was loaded in all wells. For samples, 20 ng of DNA was loaded in triplicate. Synthesized oligomers and primer sequences for qPCR in Table S2 and all samples were processed on a 96CFX real‐time system (Bio‐Rad).

High resolution melting (HRM) analysis was carried out using Precision Melt Supermix (BioRad, CA) and analyzed in a 96 CFX real time system (BioRad), using primers covering all 26 exons of the F8 gene, as described previously (Lin et al., 2008 (link)). Briefly, 2 μL of genomic DNA (12.5 ng/μL) were used for each reaction containing 5 μL of 2x Precision Melt Supermix, 2 μL primer mixture (10 pmol/μL each), and 1 μL distilled water. An initial denaturing step at 95 °C for 2 min was followed by 45 cycles of 95 °C for 10 s, 53 °C for 30 s and plate reading 72 °C for 30 s. followed by HRM with 1 cycle of 95 °C for 30 s, 1 cycle at 60 °C for 1 min, 1 cycle at 65-95 °C (10 s/step, slope 0.2 °C/s) and plate reading. HRM analysis was done with the Precision Melt analysis software (melt curve sensitivity set at 50, Tm difference threshold of 0.2 °C)