Table 1.
PCR production and sequence of primers and fluorescent probe
| Method | Gene | Forward primer (5′>3′) | Fluorescent (5′3′) | Reverse primer (5′>3′) |
|---|---|---|---|---|
| bisulfite sequencing† |
HOPX-β |
TAGTITTGTTTGGAAGAGGGGCG |
|
AACCTCCCCTAAAAACAAACTTAAC |
| Q-MSP‡ |
HOPX-β |
TTTGGAGAGGGTTTTAAAGCG |
FAM-CGGAGATAGAAGGTCGTTTATCGGGGAGGTCG-TAMRA |
AACAAACTTAACAAATCGCGAA |
| Q-MSP‡ |
β-actin |
TGGTGATGGAGGAGGTTTAGTAAGT |
FAM-ACCACCACCCAACACACAATAACAAACACA-TAMRA |
AACCAATAAAACCTACTCCTCCCTTAA |
| RT-PCR§ |
HOPX-α and γ |
CAAACCCAGGGCTTGCGCTT |
|
GCGGAGGAGCGAAACAGAGAT |
| RT-PCR§/Q-RT-PCR |
HOPX-β |
GGTCCCCCTTTCGGGAGGAA |
|
GCGGAGGAGAGAAACAGAGAT |
| RT-PCR§/Q-RT-PCR |
HOPX-core |
CAGAGGACCAGGTGGAAATCC |
|
GCGGAGGAGAGAAACAGAGAT |
| RT-PCR§/Q-RT-PCR |
β-actin |
GCTCGTCGTCGACAACGGCTC |
|
CAAACATGATCTGGGTCATCTTCT |
| PCR for cloning# | HOPX | CACCATGTCGGCGGAGACCGCGAGCGG | GTCTGTGACGGATCTGACACTCTG |
Abbreviations: Tm, annealing temperature.
†: Bisulfite sequencing PCR was done at 95°C for 3 min followed by 40 cycles at 95°C for 1 min, 72°C for 2 min, and final extension at 72°C for 10 min,in a 50 μl reaction volume containing 1 μl treated DNA, 5 μl 10× PCR buffer, 0.2 μm ol/l MgCl2, 0.2 μmol/l each primer and 0.2 μl Platinum® Taq DNA Polymerase.
‡: Q-MSP was done at 95°C for 3 min followed by 45 cycles at 95°C for 20 sec, 60°C for 30 sec, and 72°C for 30 sec, in a 25 μl reaction volume containing 100 ng treated DNA, 300 nmol/l fluorescent probe, and 25 μl iQTM supermix.
§: RT-PCR was done at 95°C for 3 min followed by 30 cycles at 95°C for 1 min, 60°C for 1 min, and final extension at 72°C for 1 min, and final extension at 72°C for 10 min, in a 50 μl reaction volume containing 1 μl cDNA, 5 μl 10× PCR buffer, 0.2 μmol/l dNTP mixture, 1.5 mmol/l MgCl2, 0.2 μm ol/l each primer and 0.2μl Platinum® Taq DNA Polymerase.
: Q-RT-PCR was done at 95°C for 2 min followed by 40 cycle at 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec, in a 25 μl reaction volume containing 100 ng treated DNA, 300 nmol/l each primer, and 25 μl iQTMPCR Mix.
#: PCR was done at 94°C for 2 min followed by 35 cycles at 94°C for 15 sec, 64.8°C for 15 sec, 68°C for 30 sec, and final extension at 68°C for 7 min, in a 50 μl reaction volume containing 1 μl cDNA, 5 μl 10× Pf×50TMPCR Mix, 0.3 μmol/l dNTP mixture, 0.3 μmol/l each primer and 1 μl Pf×50TM DNA Polymerase.