Table 4 Primers and their annealing temperatures used in Pyrosequencing
Gene Promoter | Forward Primer (5′ → 3′) [Bio] = Biotinylated at 5′ end | Reverse Primer (5′ → 3′) [Bio] = Biotinylated at 5′ end | Sequence primer (5′ → 3′) | No. of CpGs assayed | No. of CpGs Analysed | Amplicon size | Annealing temperature (°C) |
C-Kit | GGAGGGGGGAAAAAGTGTATGAAAATTTG | [Bio]TTCTACTCAATTTCTCCACCTACTT | AAATTTGGGTTTTTAGAGTAA | 8 | 8 | 179 | 63 |
Sox-2 | AGTAAGGAAGGTTTTGAGGATAGA | [Bio]ATATCATTATTCTCCCCCTCATCCACAA | AGGTTTGGGTTTTTTAAT | 6 | 4 | 187 | 64 |
Oct4 | [Bio]ATGGGGGAATTTTTTATATTTTAGAGTT | CACCACCATTAAACAAACATCC | AAAAAATTAAATAATCCCTT | 10 | 9 | 373 | 59 |
NANOG | [Bio]TGTATTTTTAGTAGAGAGGGGGTTT | ACCCAACAACAAATACTTCTAAATTCACC | ATTCACCACCTTTCCAACTT | 6 | 4 | 237 | 65 |
H19 | GGTTTTGGAGGTTAGTGTTTT | [Bio]CTCAACCCCTAAAACTAACTTAACA | TTGTATTATTTTTTTTTTT GAGAGT | 5 | 5 | 187 | 64 |