Table 1.

Primers and polymerase chain reaction (PCR) conditions for bisulphite methylation analysis

Region/gene	Primer, forward	Primer, reverse	PCR conditions
Xist, region 1A – 1st round	MM3, gttaattaatgtagaagaatttttagtgttta	MM2, aaatattcccccaaaactccttaaataa	94°C for 15 minutes; (94°C for 20 seconds; 54°C for 30 seconds; 72°C for 90 seconds) × 25 cycles; 72°C for 5 minutes
Xist, region 1A – nested	Tag 6F, aggaagagagttgtaaaataggataattttttattat	T7 6R, cagtaatacgactcactatagggagaaggctatattcccccaaaactccttaaata	94°C for 15 minutes; (94°C for 20 seconds; 54°C for 30 seconds; 72°C for 30 seconds) × 5 cycles; (94°C for 20 seconds; 60°C for 30 seconds; 72°C for 30 seconds) × 25 cycles; 72°C for 5 minutes
Xist, region 2C	Tag 1F, aggaagagagattttgtttttaaattgagtgggtg	T7 1R, cagtaatacgactcactatagggagaaggcttaaaccctatcccctaatcctctac	94°C for 15 minutes; (94°C for 20 seconds; 60°C for 30 seconds; 72°C for 1 minute) × 5 cycles; (94°C for 20 seconds; 68°C for 30 minutes; 72°C for 1 minute) × 40 cycles; 72°C for 5 minutes
Xist, region 2D	Tag 5F, aggaagagagagggtgtgtgtgtatatggattttt	T7 MM5, cagtaatacgactcactatagggagaaggcttaactcaaacataaaactaacatataacaca	94°C for 15 minutes; (94°C for 20 seconds; 54°C for 30 seconds; 72°C for 1 minute) × 5 cycles; (94°C for 20 seconds; 58°C for 30 seconds; 72°C for 1 minute) × 40 cycles; 72°C for 5 minutes
Igf2r	Tag Igf2r_F2, aggaagagaggtgtggtatttttatgtatagttagg	T7 Igf2r_R cagtaatacgactcactatagggagaaggctaaatatcctaaaaatacaaactacac	94°C for 15 minutes; (94°C for 20 seconds; 54°C for 30 seconds; 72°C for 1 minute) × 45 cycles; 72°C for 5 minutes
H19A	Tag H19A, aggaagagaggagaaaatagttattgtttatagtttt	T7 H19A, cagtaatacgactcactatagggagaaggctcctaaaatactaaacttaaataacccacaa	94°C for 15 minutes; (94°C for 20 seconds; 50°C for 30 seconds; 72°C for 1 minute) × 45 cycles; 72°C for 5 minutes
H19B	Tag H19A, aggaagagagttagtgtggtttattataggaaggtatagaagt	T7 H19A, cagtaatacgactcactatagggagaaggct taaacctaaaatactcaaactttatcacaac	94°C for 15 minutes; (94°C for 20 seconds; 50°C for 30 seconds; 72°C for 1 minute) × 5 cycles; (94°C for 20 seconds; 58°C for 30 seconds; 72°C for 1 minute) × 40 cycles; 72°C for 5 minutes