Table 2

PCR primer sequences for SSCP, bisulfite sequencing, and MS-PCR assay

PrimerSequenceProduct sizeAnnealing temperatureCycles
SSCP
 Exon 1
  RAE1-A15′-CCTGCTAGCGCCCAAAGC-3′8355°C35
  RAE1-A25′-CAGCTCCCGCAGCTCAAT-3′
  RAE1-B15′-ATGTCGGGGGAGCCTGAG-3′15158°C35
  RAE1-B25′-CACGGCCAGGGACCAG-3′
  RAE1-C15′-CTGGAGCGTGCCAACG-3′15055°C35
  RAE1-C25′-GACGCCCCAGATGAAG-3′
  RAE1-D15′-CACACGGCAGCTGGTC-3′8758°C35
  RAE1-D25′-AAGTCGCCACAGAGGTC-3′
 Exon 2
  RAE2-15′-GGGACATTTCCCCGACCT-3′20055°C35
  RAE2-25′-ATCCTCGCCCTTCCCATAC-3′
 Exon 3
  RAE3-15′-CCAGAGGCCATTTTTCAGAG-3′22655°C35
  RAE3-25′-TAGCTGGGTACCTGCTCCTC-3′
 Exon 4
  RAE4-A15′-ACTGACATCTCACCTGGAACC-3′24055°C35
  RAE4-A25′-CACATGCAGGTGCTTGACA-3′
  RAE4-B15′-GCCGCACTTCCTTTTACCT-3′21055°C35
  RAE4-B25′-GCATGCGTATATACCCTCACA-3′
 Exon 5
  RAE5-15′-TGGCCCTGTCTCTGATCATT-3′23155°C35
  RAE5-25′-TCCTCCTCCAAGCCTTACTG-3′
 Exon 6
  RAE6-B15′-CCTGTACACTCCCCTTTTGC-3′19458°C35
  RAE6-B25′-ACCTGGGGGTACAAGAGGTC-3′
Bisulfite sequencing
 1st PCR
  RABS-15′-GTTTTGGTAGTTTAATGAGTTTAGGTTTTTT-3′55°C30
  RABS-35′-CCCCACAATCCCTACACCCAAAT-3′
 2nd PCR
  RABS-25′-ACCCTCTTCCTCTAACACAATAAAACTAACC-3′20555°C30
  RABS-35′-CCCCACAATCCCTACACCCAAAT-3′
Methylated specific PCR Assay
 Methylated
  RAM-15′-GTGTTAACGCGTTGCGTATC-3′9360°C35
  RAM-25′-AACCCCGCGAACTAAAAACGA-3′
 Unmethylated
  RAU-15′-TTTGGTTGGAGTGTGTTAATGTG-3′10560°C35
  RAU-25′-CAAACCCCACAAACTAAAAACAA-3′
RT-PCR
 1st PCR
  RTE1F5′-ACACGTGGTGCGACCTCT-3′65°C35
  RTE4R5′-GATGAAGCCTGTGTAAGAACCGTCCT-3′
 2nd PCR
  RTE2F5′-CAGATTGCAAGTTCACCTGCCACTA-3′24265°C25
  RTE4R5′-GATGAAGCCTGTGTAAGAACCGTCCT-3′