Table 1 Primers and amplification conditions for PCR, pyrosequencing, and RT-qPCR experiments
Gene/region | Assay | Primers | PCR Conditions |
|---|---|---|---|
IFNγ promoter CpG -186 | PCR and Pyrosequencing | F: 5’-biotin-AGATGGTGATAGATAGGTAGGGATGATA-3’ | 95°C, 15 min; 45 cycles of 95°C, 30 sec; 55°C, 30 sec; 72°C, 30 sec; 72°C, 10 min; 4°C hold |
R: 5’-TCCCACCAAAATAACACAAATAAACAT -3’ | |||
S: 5’-AAATAAACATAATAAATCTATCTCA-3’ | |||
IFNγ promoter CpG -54 | F: 5’ATGTGTTGTATTTTTTTTGGTTGTTGGTAT-3’ | ||
R: 5’-biotin-TATCATCCCTACCTATCTATCACCATCTC-3’ | |||
S: 5’-ATTGAAGTTTTTTGAGGATT-3’ | |||
IFNγ promoter (target) | RT-qPCR | F: 5’-TCGGTAACTGACTTGAATGTCCA-3’ | 95°C, 3 min; 40 cycles of 90°C, 10 sec; 55°C, 30 sec; 4°C hold |
R: 5’-TCGCTTCCCTGTTTTAGCTGC-3’ | |||
GAPDH (internal control) | F: 5’-ACAACTTTGGTATCGTGGAAGG-3’ | ||
R: 5’-GCCATCACGCCACAGTTTC-3’ |