Skip to main content

Table 1 Primers and polymerase chain reaction (PCR) conditions for bisulphite methylation analysis

From: Dicer regulates Xist promoter methylation in ES cells indirectly through transcriptional control of Dnmt3a

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, aggaagagag ttgtaaaataggataattttttattat

T7 6R, cagtaatacgactcactatagggagaaggct atattcccccaaaactccttaaata

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, aggaagagag attttgtttttaaattgagtgggtg

T7 1R, cagtaatacgactcactatagggagaaggct taaaccctatcccctaatcctctac

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, aggaagagag agggtgtgtgtgtatatggattttt

T7 MM5, cagtaatacgactcactatagggagaaggct taactcaaacataaaactaacatataacaca

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, aggaagagag gtgtggtatttttatgtatagttagg

T7 Igf2r_R cagtaatacgactcactatagggagaaggct aaatatcctaaaaatacaaactacac

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, aggaagagag gagaaaatagttattgtttatagtttt

T7 H19A, cagtaatacgactcactatagggagaaggct cctaaaatactaaacttaaataacccacaa

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, aggaagagag ttagtgtggtttattataggaaggtatagaagt

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