Abi methyl primer express v1

The HHIP BSP primer was designed by ABI Methyl Primer Express v1.0 software (Applied Biosystems). The primer sequences of HHIP were as follows: forward, 5′-GGGGAGGAGAGAGGAGTTTG-3′ and reverse, 5′-CCCCACCACCTCCCTACTAC-3′, with an amplified fragment length of 243 bp. The primer sequences of the internal control, β-actin, were as follows: forward, 5′-AGAGCTACGAGCTGCCTGAC-3′ and reverse, 5′-AGCACTGTGTTGGCGTACAG-3′, with an amplified fragment length of 184 bp. The volume of the reaction agents was 50 μl, with reaction conditions of 94°C for 30 sec, 60°C for 40 sec and 72°C for 50 sec. The BSP products were sent to Shanghai Shengong Biological Engineering Co., Ltd. (Shanghai, China) for sequence analysis.

The HHIP MSP primer was designed by ABI Methyl Primer Express v1.0 software (Applied Biosystems, Foster City, CA, USA). The methylation primer sequences of HHIP were as follows: forward, 5′-GTAGTAGTCGGGTAGTTTCGGAATTTTC-3′ and reverse, 5′-AAAAACGACTAACCGCGACG-3′, with an amplified fragment length of 190 bp. The non-methylation primer sequences were as follows: forward, 5′-AGTAGTTGGGTAGTTTTGGAATTTTTGG-3′ and reverse, 5′-AAAAACAACTAACCACAACA-3′, with an amplified fragment length of 188 bp. The volume of the reaction agents was 50 μl, with reaction conditions of 94°C for 30 sec, 60°C for 40 sec and 72°C for 50 sec. The PCR products were subjected to 1.5% agarose gel electrophoresis analysis.