Quantification of Cell-Free DNA Integrity

The isolated cfDNA was subjected to quantitative real-time PCR, which used two different primer sets to amplify both shorter (ALU115) and longer (ALU247) fragments of consensus ALU sequences. Previously published primer sequences were used to amplify both ALU115 and ALU247 fragments (13 (link)). The standard PCR reaction mixture in each well contained 10μL iQ SYBR Green Supermix (Bio-Rad), 1 μL each of forward and reverse primers, 1 μL isolated cell-free DNA and 7 μL of RNase free water for a total reaction volume of 20 μL. The samples were run along with standards on the StepOnePlus Real-Time PCR Detection System (Applied Biosystems). The Human genomic DNA (G3041, Promega) was used as a standard to determine ALU247 and ALU115 amplicons concentration. The absolute concentration of ALU247 and ALU115 fragments in each sample was determined from the standard curve (Supplemental Figure 1). The assay was repeated twice in two technical replicates and the cell-free DNA integrity index (cfDI) was calculated as the ratio of the concentration of ALU247 fragments to ALU115 fragments.