The second step consisted of 18 cycles under the same cycling conditions, with 5 sec added to the elongation step in each cycle.
Three microliters of RT-PCR reaction were added to the second step (nested) PCR and amplified for other 30 cycles, using the same cycling conditions of the first step.
The same cycling conditions were used throughout; 30 cycles at 94°C for 18 s, 50°C for 90 s and 72°C for 30 s, and a terminal 72°C elongation step for 300 s.
A volume of 2 μl of the mPCR product was used as the template of each sPCR, and the same cycling conditions were used for 45 cycles.
When the PCR program reached the lower annealing temperature of 52°C, 28 additional cycles were performed by using the same cycling conditions except for the use of a constant annealing temperature of 52°C.
PCR amplification was carried out in similar conditions (2 pmol of each primer, 1× Qiagen multiplex mastermix, 0.5× Q-Solution, and approximately 30 ng of DNA in a total volume of 10µl) and the same cycling profile (15 min at 95°C, followed by 30 cycles 30 s at 94°C, 90 s at 56°C and 60 s at 72°C and a final extension for 10 min) for all markers, using a commercial multiplex PCR kit (Qiagen).
The nested PCR used the same cycling parameters for the second round, but the number of cycles was increased to 40.
Gtl2 IG-DMR, with the first nucleotide of IG-BS-F1 corresponding to position 110,766,235: 30 cycles of 94°C for 30 s, 52°C for 1 min, 72°C for 1 min, using primers IG-BS-F1/IG-BS-R, followed by 35 cycles using IG-BS-F2/IG-BS-R and the same cycling conditions as above.
All reactions were optimized to obtain the best amplification kinetics under the same cycling conditions (denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 60 s) and composition of the reaction mixture.
Both rounds of the PCR were carried out under the same cycling conditions: an initial denaturation stage at 95°C for 2 minutes; 35 cycles consisting of a denaturation stage at 95°C for 20 seconds, an annealing stage at 65°C for 20 seconds and an extension stage at 72°C for 1 minute; and a final extension stage at 72°C for 5 minutes.
The same cycling parameters were used in the second PCR with an increase to 40 cycles.
