Effects of DNA methylation on expression of tumor suppressor genes and proto-oncogene in human colon cancer cell lines

Table 1 Sequences of primers and program of PCR

Primers	Sense(5’→3’)	Antisense(5’→3’)	Size of product and PCR condition	GenBank accession number
β-actin RT-PCR	GGAGTCCTG	CTAGAAGCA	322 bp	XM004814
(for p16INK4A RT-PCR in HT-29)	TGGCATCCACG	TTTGCGGTGGA	94 °C 3 m; 94 °C 30 s, 60 °C 1 m, 72 °C 1 m, 27 X; 72 °C 5 m
β-actin RT-PCR	GGCATCGTG	GCTGGAAGG	612 bp	BC023204
(for RT-PCR in other cells)	ATGGACTCCG	TGGACAGCGA	94 °C 5 min; 92 °C 40 s, 58 °C 40 s, 72 °C 50 s, 30 X; 72 °C 5 min
p16INK4A	CCCGCTTTC	TTATTTGAG	355 bp	L27211
RT-PCR	GTAGTTTTCAT	CTTTGGTTCTG	94 °C 5 min; 94 °C 1 min, 58 °C 1 min, 72 °C, 1 min, 35 X; 72 °C 5 min
APC	GAGACAGAA	GTAAGATGATTG	170 bp	AF209032
RT-PCR	TGGAGGTGCTGC	GAATTATCTTCTA	95 °C 5 min; 95 °C 1 min, 53 °C 1 min, 72 °C, 1 min, 35 X; 72 °C 5 min
p21WAF1	CAGGGGACA	GGGCGGCCA	335 bp	NM_000389
RT-PCR	GCAGAGGAAGA	GGGTATGTAC	94 °C 5 min; 94 °C 1 min, 58 °C 1 min, 72 °C 1 min, 35 X; 72 °C 5 min
c-myc	CCAACAGGA	CTCGGTCACCAT	290 bp	V00568
RT-PCR	GCTATGACCTC	CTCCAGCT	94 °C 5 min; 94 °C 1 min, 52 °C 1 min, 72 °C, 1 min, 35 X; 72 °C 5 min
p16INK4A MSP	CAGAGGGTG	CGGGCCGCG	140 bp	X94154
(Wild-type)	GGGCGGACCCGC	GCCGTGG	95 °C 5 min; 95 °C 1 min, 65 °C 2 min, 72 °C 3 min, 5 X; 95 °C 30 s, 65 °C 30 s, 72 °C 1 min, 35 X; 72 °C 5 min
p16INK4AMS	TTATTAGAGGGT	GACCCCGAACCG	150 bp	X94154
P-methyl-primers	GGGGCGGATCGC	CGACCGTAA	95 °C 5 min; 95 °C 1 min, 65 °C 2 min, 72 °C 3 min, 5 X; 95 °C 30 s, 65 °C 30 s, 72 °C 1 min, 35 X; 72 °C 5 min
p16INK4A	TTATTAGAGGGT	CAACCCCAAACC	151 bp	X94154
MSP-unmethyl primers	GGGGTGGATTGT	ACAACCATAA	95 °C 5 min; 95 °C 1 min, 60 °C 2 min, 72 °C 3 m °C, 5 X; 95 °C 30 s, 60 °C 30 s, 72 °C 1 min, 35 X; 70 °C 5 min

Table 2 The expression of p16^INK4A gene in HT-29 cells (the band density)

5-aza-dC conc	Mock treated	1 μM, 24 h	10 μM, 24 h
Density	2257.7	2782.5	1975.3

The density of each band from RT-PCR in each lane of Figure 2 was normalized to the amount of total RNA as determined by the density of band in RT-PCR for β-actin.

Table 3 Expression of p16^INK4A gene in SW1116 and Colo-320 cells (the band density)

5-aza-dC treatment	Mock treated	2 μmol/L, 24 h	5 μmol/L, 24 h	10 μmol/L, 24 h	2 μmol/L, 72 h	5 μmol/L, 72 h	10 μmol/L, 24 h
SW1116	1494.7	2055.5	2436.9	3487.3	1592.0	2074.8	2774.0
Colo-320	809.1	860.6	829.2	1298.8	875.7	923.5	1189.6

The density of each band from RT-PCR in each lane of Figure 3 was normalized to the amount of total RNA as determined by the density of band in RT-PCR for β-actin.

Table 4 Expression of APC gene in SW1116 and Colo-320 cells (the band density)

5-aza-dC treatment	Mock treated	2 μmol/L, 24 h	5 μmol/L, 24 h	10 μmol/L, 24 h	2 μmol/L, 72 h	5 μmol/L, 72 h	10 μmol/L, 24 h
SW1116	786.2	1481.2	782.6	796.9	802.9	1173.5	1236.8
Colo-320	1804.6	2388.2	4055.2	1923.9	1803.0	3197.8	3271.7

The density of each band from RT-PCR in each lane of Figure.4 was normalized to the amount of total RNA as determined by the density of band in RT-PCR for β-actin.