Supplementary Fig. 1. Illustrations of 9 tested miR-hosting CpG islands. Open frame, location of pre-miRNA
transcript. Open arrow, pre-miRNA transcript direction; Blue-underline, locations of the forward and reversed
primer-matching regions used to detect methylation of CpG island with DHPLC and bisulfite sequencing;
Purple vertical bars, CpG sites
1
m
8V
U
7
H2O
B
6
GES-1
M
CALU3
5
HCT116
H1299
4
MGC803
SGC7901
3
DU145
HEPG2
2
AGS
MKN74
1
SIHA
MB
0
3
4
5
min
Supplementary Fig. 2. DHPLC chromatogram of methylated and unmethylated miR-9-1 in various cell lines.
UV-detector; the partial denaturing temperature, 55.4C; U, peak for the unmethylated PCR products; M, peak
for the methylated PCR products; B, peripheral blood DNA; MB, M.sssI-methylated blood DNA
2
mV
10
U
U
H2O
B
H1299
SIHA
M
A549
DU145
SGC7901
BGC823
MKN45
RKO
HELA
MGC803
HEPG2
AGS
MKN74
MB
0
3
4
5
min
Supplementary Fig. 3. DHPLC chromatogram of methylated and unmethylated miR-9-3 in various cell lines.
UV-detector; the partial denaturing temperature, 58.5C; U, peak for the unmethylated PCR products; M, peak
for the methylated PCR products; B, peripheral blood DNA; MB, M.sssI-methylated blood DNA
3
mV
U
8
7
H2O
B
6
PG
M (partial)
GES-1
A549
5
CALU3
4
MKN45
M (full)
HCT116
3
RKO
MGC803
2
BGC823
AGS
293T
1
MB
2
3
4
5
min
Supplementary Fig. 4. DHPLC chromatogram of methylated and unmethylated miR-34b in various cell lines.
UV-detector; the partial denaturing temperature, 56.8C; U, peak for the unmethylated PCR products; M
(partial or full), peak for the partially or fully methylated PCR products; B, peripheral blood DNA; MB,
M.sssI-methylated blood DNA
4
200
mV
U
H2O
B
MKN45
AGS
100
HELA
SIHA
DU145
RKO
A549
M
MGC803
PC-3
KG1A
HL60
MB
0
2
3
4
5
min
Supplementary Fig. 5. DHPLC chromatogram of methylated and unmethylated miR-210 in various cell lines.
Fluorescence-detector; the partial denaturing temperature, 58.7C; U, peak for the unmethylated PCR
products; M, peak for the methylated PCR products; B, peripheral blood DNA; MB, M.sssI-methylated blood
DNA
5
U
mV
900
H2O
B
800
H1299
A549
700
SGC7901
M
SIHA
600
MGC803
HEPG2
500
293T
MKN45
400
BGC823
HELA
300
SW480
HCT116
200
AGS
MKN74
100
RKO
MB
0
2
3
4
5
min
Supplementary Fig. 6. DHPLC chromatogram of methylated and unmethylated miR-137 in various cell lines.
Fluorescence-detector; the partial denaturing temperature, 58.3C; U, peak for the unmethylated PCR
products; M, peak for the methylated PCR products; B, peripheral blood DNA; MB, M.sssI-methylated blood
DNA
6
mV
U
H2O
B
CALU3
SW480
M
GES-1
PC-3
H1299
MGC803
A549
RKO
HELA
BGC823
SGC7901
AGS
MKN45
293T
MKN74
MB
0
3
4
5
min
Supplementary Fig. 7. DHPLC chromatogram of methylated and unmethylated miR-375 in various cell lines.
UV-detector; the partial denaturing temperature, 55.7C; U, peak for the unmethylated PCR products; M, peak
for the methylated PCR products; B, peripheral blood DNA; MB, M.sssI-methylated blood DNA
7
mV
U
H2O
10
M
DU145
HCT116
SW480
MKN74
SIHA
A549
HELA
BGC823
GES-1
0
SGC7901
MGC803
H1299
HL60
KG1A
MKN45
MB
B
-10
3
4
5
min
Supplementary Fig. 8. DHPLC chromatogram of methylated and unmethylated miR-200b in various cell
lines. UV-detector; the partial denaturing temperature, 55.6C; U, peak for the unmethylated PCR products;
M, peak for the methylated PCR products; B, peripheral blood DNA; MB, M.sssI-methylated blood DNA
8
mV
U
H2O
70
B
60
M
F0856
50
F1297
40
F1047
30
MKN74
20
AGS
10
MB
0
3
4
5
min
Supplementary Fig. 9. DHPLC chromatogram of methylated and unmethylated miR-193b in various cell
lines. Fluorescence-detector; the partial denaturing temperature, 56.5C; U, peak for the unmethylated PCR
products; M, peak for the methylated PCR products; B, peripheral blood DNA; MB, M.sssI-methylated blood
DNA
9
U
mV
M
6
5
4
AGS
3
MKN74
MGC803
2
SGC7901
BGC823
1
MKN45
H2O
2
3
4
5
min
Supplementary Fig. 10. DHPLC chromatogram of methylated and unmethylated miR-203 in various cell
lines. UV-detector; the partial denaturing temperature, 57.7C; U, peak for the unmethylated PCR products;
M, peak for the methylated PCR products; B, peripheral blood DNA; MB, M.sssI-methylated blood DNA
10
Supplementary Fig. 11. Distribution of miR methylation in various gastric mucosa samples with and without
H. pylori infection. Each line represents methylation status of one miR gene and each column represents one
sample. The proportion of methylated allele is displayed step-wisely: negative (white), positive and <30%
(light gray), or 30%-60% (deep gray) or >60% (black). Not informative sample is marked as “ND”.
The case with or without H. pylori-specific 23S rDNA was labeled with letter “p” (light green) and “n”
(blue).
11
Supplementary Fig. 12. Comparison of levels of 7 mature miRNAs by quantitative RT-PCR in 20 pairs of
fresh gastric carcinoma (GC) and the corresponding surgical margin (SM) tissue samples. The relative miRNA
levels of the paired GC and SM samples from the same patient were linked by a line.
12