Table 1.
Oligonucleotide DNA Used in This Study
| Name | Forward Primer (5′‐3′) | Reverse Primer (5′‐3′) | Footnotes |
|---|---|---|---|
| Real‐time reverse transcriptase PCR | |||
| GAPDH | tcattgacctcaactacatggttt | ttgattttggagggatctcg | Human |
| ACTB | tggcacccagcacaatgaa | ctaagtcatagtccgcctagaagca | Human |
| Rn18s | acggaagggcaccaccagga | caccaccacccacggaatcg | Rat |
| Bisulfite sequencing | |||
| Human CYP11B2 BM (−290 to +11) | gaaaggagaggttaggttttattatttt | accctttacaaaaacaaccaaaa | CpG1 to CpG3 |
| Human CYP11B2 BM (−278 to +69) | gaaaggagaggttaggttttattatttt | accctttacaaaaacaaccaaaa | CpG‐2 to CpG3 |
| Rat CYP11B2BM | ctcagacaagtccagggacagtttc | ggcaacactggcccccaagacactg | CpG5 to CpG8 |
| Luciferase assay | |||
| CYP11B2 (−167 KpnI/+9 NheI) | atggggtaccggcacctgacttctccttca | Atcggctagcattccaatgctccctccac | |
| CYP11B2 (−475 KpnI/+9 NheI) | atggggtaccaaccagtgcgctccgt | Atcggctagcattccaatgctccctccac | |
| pGL4.10 BM | ttttagtgtaagtgtaggtg | aaccaaatcttaatatcct | |
| Name | Biotinylated Double‐Stranded Oligonucleotide (5′‐3′) | ||
|---|---|---|---|
| NoShift assay | |||
| Unmethylated Ad1 (CpG1) | agccagttctcccatgacgtgatatgtttccag‐biotin | The bold Cs indicate cytosine residues methylated in vitro. | |
| Methylated Ad1 (CpG1) | agccagttctcccatgaCgtgatatgtttccag‐biotin | ||
| Unmethylated Ad5 (CpG2) | ggctgggggcctccagccttgaccttcgctctgaga‐biotin | ||
| Methylated Ad5 (CpG2) | ggctgggggcctccagccttgaccttCgctctgaga‐biotin | ||
| Unmethylated CpG3 | gtccagaccccacgccttttct‐biotin | ||
| Methylated CpG3 | gtccagaccccaCgccttttct‐biotin | ||
| Chromatin immunoprecipitation‐quantitative PCR | |||
| R1 (Ad1/CpG1) | gagtctcaggcaggcaggtccaga | attccaatgctccctccac | 113 bp |
| R2 (Ad5/CpG2) | ggcacctgacttctccttca | gagaactggctctggacctg | 93 bp |
| R3 (CpG3) | gagaaaggagaggccaggtc | gtcctgctggtgtgaggatg | 94 bp |
| R4 | aaaaagaaaccaggggttgg | ttgcttgcatggtttctgag | 95 bp |
| GNAS CpG island region | ttctcgtccgaagatacgaaa | gccgaggtaggtgtcagaga | 76 bp |
| R1 (Luc‐Ad1) | gagtctcaggcaggtccaga | cttaatgtttttggcatcttcca | |
| R2 (Luc‐Ad5) | ctagcaaaataggctgtccc | gagaactggctcggacctg | |
| Name | Sequencing Primer | Footnotes |
|---|---|---|
| Pyrosequencing | ||
| Rat CYP11B2(1) | aagagtaggtgtagtg | CpG2 to 4CpG2 to 7CpG2 to 10 |
| Rat CYP11B2(2) | ggaagaaattgtttttgtg | |
| Rat CYP11B2(3) | tgttttgggggttag | |
| Name | Forward Primer (5′‐3′) | Reverse Primer (5′‐3′) (Biotinylated) | Footnotes |
|---|---|---|---|
| Pyrosequencing | |||
| Rat CYP11B2(1) | tttgagttttggttatggttggggtttaat | aaaaaataatttcttccccttctacct | CpG2 to 4 |
| Rat CYP11B2(2) | gtttttggagtattgtggtatgg | ttctaccccccaacaaactttac | CpG2 to 7 |
| Rat CYP11B2(3) | gttatttgtttttggagtattgtggta | actaacaattaaaccccaaccat | CpG2 to 10 |
ACTD indicates beta actin; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PCR, polymerase chain reaction; Rn18, 18s ribosomal RNA.