Quantitative PCR Protocol for Genomic DNA Analysis

For qPCR methods, see the work of Koh and Murray (53 (link)). Briefly, the QIAgility (Qiagen) robot and software were used for setting up reactions. The Luna Universal qPCR master mix (NEB) was used in reactions, utilizing SYBR green dye-based detection and quantitation. Each 20-μl reaction mixture contained 10 μl qPCR master mix and 2 μl each 10 μM primer and ∼120 ng genomic DNA. Every reaction was set up in triplicate, and the final data were summarized as a mean average. The Rotor-Gene Q PCR instrument (Qiagen) was used to carry out reactions, with the accompanying software used to analyze results. The PCR was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of the two-step reaction of 95°C for 5 s and 60°C for 30 s. Following the 40 cycles, the temperature was increased from 60°C to 95°C in 1°C increments every 5 s in order to produce melt curves. From the fluorescent amplification curves that were produced from the 40 cycles, threshold cycle (C_T) values were calculated. The C_T values were processed using the calculation 1/(2^CT) to make the resulting values more biologically relevant.