Table 1.

Alternative incubation protocols tested to evaluate the performance ofthe bisulfite treatment kits

Name of the protocol	Incubation steps
Cycling 16 h	16 cycles of 95° C for 30 s and 50° C for 1 h, hold at 4° C
Cycling 8 h	95°C for 5 s, 60° C for 30 s, 8 cycles of 95° C for 6 s and 60° C for 30 s, hold at 4° C
Fixed 3 h	98°C for l0 s and 64° C for 2 h 40 min, hold at 4° C

All five kits included in this study [Epitect+ (Qiagen], MethylDetector (Active Motif), Zymo Gold, Zymo Methylation and Zymo Lightning (all three from Zymo Research]] were tested according to the protocol specified by the respective manufacturer for each kit. In addition, the Epitect+, MethylDetector, Zymo Gold and Zymo Methylation were tested using the protocols herein listed to evaluate the influence of type and duration ofDNA incubation ontheir performance.