Table 4 Primer sequences for IGFBP-3 analysis.
From: Promoter methylation of IGFBP-3 and p53 expression in ovarian endometrioid carcinoma
Annealing °C (Cycle) | Size (bp) | ||
|---|---|---|---|
Quantitative real time methylation-specific PCR | |||
IGFBP3 | F: 5'- AGGTGATTCGGGTTTCGGGC -3' R: 5'- GACCCGAACGCGCCG -3' | 60 (40) | 223 |
Methylation-specific PCR | |||
IGFBP3 _MSP | F: 5'-TCGGGTATATTTTGGTTTTTGTAG-3' R: 5'-AAACATATAAAATCCAAACAAAAA-3' | 55 (30) | 351 |
IGFBP3 _M (methythlated) | F: 5'CGAAGTACGGGTTTCGTAGTCG-3' R: 5'-CGAC CCGAACGCGCCGACC-3' | 66 (40) | 129 |
IGFBP3 _U (unmethylated) | F: 5'-TTGGTTGTTTAGGGTGAAGTATGGGT-3' R: 5'-CACCCAACCACAATACTCACATC-3' | 64 (40) | 158 |
Bisulphate-PCR | |||
IGFBP3 | F: 5'-TTTGAGAGTGGAAGGGGTAAGGG-3' R: 5'-CCCACTACATAACACCTACAACC-3' | 53 (40) | 512 |
Mutagenesis | |||
Wild type IGFBP3 | F: 5'-GGGCACACCTTGGTTCTTGTAG-3' R: 5'-TTCCTGCCTGGATTCCACAGCT-3' | 52 (40) | 316 |
Mutant (M12) IGFBP3 | F: 5'-ACAAGGTGACCCGGGCTCAGGGAGTGAGCACGAGGAGCAGGT-3' R: 5'-ACCTGCTCCTCGTGCTCACTCCCTGAGCCCGGGTCACCTTGT-3' | 55 (12) | |
Mutant (M34) IGFBP3 | F: 5'-GCACGAGGAGACGGTGCCAGGGAGAGTCTCAAGCTCCACGCC-3' R: 5'-GGCGTGCAGCTTGAGACTCTCCCTGGCACCTGCTCCTCGTGC-3' | 55 (12) | |