Table 3

Determining sensitivity of quantitative multiplexed methylation-specific PCR

Methylationa (%)
RASSF1ATWISTHIN1Cyclin D2
Copies Mb and U templatec,d
 1000/100,0000.92202.76044.80241.5760
 100/100,0000.06020.24820.62730.1525
 10/100,0000.00780.02720.00240.0025
 1/100,0000.00720.00120.00000.0000
 0.1/100,0000.00000.00000.00000.0000
HSD (100% U control)0.00000.00030.00000.0000
231 (100% M control)99.9981100.0000100.0000100.0000
Water0.00000.00000.00000.0000
Copies M and Uc,e
 300/29,7000.78231.2190
 30/2,9700.71950.9376
 3/2970.23900.1451
  • a The percentages of methylation of RASSF1A, TWIST, HIN1, and Cyclin D2 from the quantitative multiplexed methylation-specific PCR assay are shown. The assay sensitivity is 1–10 in 100,000 for methylated DNA, and it detects as few as 1–3 copies of methylated DNA.

  • b M, methylated; U, unmethylated; HSD, human sperm DNA; 231, MDA-MB231.

  • c Purified methylated and unmethylated stock DNA template were mixed in the proportions indicated.

  • d Unmethylated DNA was kept constant as the amount of methylated DNA was decreased.

  • e A 1% M control was kept constant as the total quantity of DNA decreased.