Background
Methods
Samples
DNA extraction from frozen tissues: Nucleospin®96 tissue kit (Macherey Nagel®)
DNA extraction from formalin-fixed paraffin embedded (FFPE) tissues
Classical method
Dedicated Method to FFPE tissue: QIAamp® DNA FFPE Tissue kit (QIAGEN®)
Quantification and quality assessment of DNA
DNA bisulfite conversion - EpiTect Bisulfite kit (QIAGEN®)
PCR and pyrosequencing assays
Measurement of LINE-1 methylation level
Measurement of MLH1 and MGMT methylation levels
Assessment of reproducibility of bisulfite conversion, PCR/Pyrosequencing
Results
Variability of measurement of the methylation level of DNA from cryo-preserved tissue
converted DNA samples | PCR 1 (% of methylation) | PCR 2 (% of methylation) | SD due to different PCR and different pyrosequencing | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
pyrosequencing 1 | pyrosequencing 2 | pyrosequencing 1 | pyrosequencing 2 | |||||||||||||
CpG site 1 | CpG site 2 | CpG site 3 | CpG site 1 | CpG site 2 | CpG site 3 | CpG site 1 | CpG site 2 | CpG site 3 | CpG site 1 | CpG site 2 | CpG site 3 | CpG site 1 | CpG site 2 | CpG site 3 | ||
A1 | 55.9 | 62.9 | 62.3 | 55.0 | 62.1 | 62.2 | 56.5 | 61.6 | 59.3 | 54.6 | 63.4 | 61.4 | 0.9 | 0.8 | 1.4 | mean = 0.9 |
A2 |
53.1
| 63.1 | 59.7 | 56.0 | 63.3 | 59.5 | 53.2 | 62.7 | 60.0 | 57.5 | 62.3 | 60.5 | 2.2 | 0.4 | 0.4 | |
A3 | 55.7 | 61.9 |
58.6
| 55.4 | 61.8 | 61.1 | 54.8 |
60.6
| 59.6 | 55.3 | 62.5 | 61.6 | 0.4 | 0.8 | 1.4 | |
A4 | 54.1 | 63.8 | 60.7 | 56.4 | 63.6 | 59.9 | 56.4 | 63.7 | 60.3 | 55.8 | 64.1 | 59.6 | 1.1 | 0.2 | 0.5 | |
B1 | 56.3 | 61.8 | 62.7 | 57.8 | 63.1 |
63.5
| 58.0 | 62.9 | 60.6 | 56.7 | 64.1 | 61.4 | 0.8 | 0.9 | 1.3 | |
B2 | 55.9 | 63.4 | 62.9 | 57.9 | 64.0 | 63.2 | 55.2 | 63.1 | 62.5 |
58.6
|
64.7
| 63.3 | 1.6 | 0.7 | 0.4 | |
bisulfite-to-bisulfite SD | 1.3 | 0.8 | 1.8 | 1.2 | 0.9 | 1.7 | 1.7 | 1.1 | 1.1 | 1.5 | 0.9 | 1.2 | ||||
mean = 1.3 | ||||||||||||||||
SD between all measures of site 1 | 1.4% | |||||||||||||||
SD between all measures of site 2 | 1.0% | |||||||||||||||
SD between all measures of site 3 | 1.4% |
Qualitative analysis of bisulfite conversion for FFPE tissues
Classical extraction method
Dedicated extraction kit for the LINE-1 marker
Dedicated extraction kit for MLH1 and MGMT
Qualitative analysis of bisulfite conversion for cryo-preserved tissues
Identity | FFPE DNA 1 | FFPE DNA 2 | FFPE DNA 2 | FFPE DNA 2 | Cryo. DNA | Cryo. DNA | Cryo. DNA | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
LINE-1
|
LINE-1
|
MLH1
|
MGMT
|
LINE-1
|
MLH1
|
MGMT
| ||||||||
PCR 1 | PCR 2 | PCR 1 | PCR 2 | PCR 1 | PCR 2 | PCR 1 | PCR 2 | PCR 1 | PCR 2 | PCR 1 | PCR 2 | PCR 1 | PCR 2 | |
1 |
check
|
failed
| passed | passed | passed | passed | passed | passed | passed | passed | ||||
2 |
failed
| passed |
check
| passed |
⁄
| passed | passed | passed | passed | passed | ||||
3 | passed | passed |
check
|
check
|
⁄
|
failed
|
failed
| passed | passed | passed | passed | |||
4 |
failed
|
failed
| passed |
check
|
⁄
|
failed
| passed | passed | passed | passed | passed | |||
5 |
failed
| passed |
failed
|
check
|
⁄
| passed | passed | passed | passed | passed | passed | passed | ||
6 |
check
| passed | passed | passed | passed | passed | passed | passed | passed | passed | ||||
7 |
failed
|
failed
|
failed
|
check
| passed | passed |
failed
| passed | passed | passed | passed |
check
|
check
| |
8 | passed |
failed
| passed | passed | passed | passed | passed | passed | passed | passed | ||||
9 |
check
| passed | passed | passed | passed | passed | passed | passed | passed | passed | ||||
10 |
failed
|
failed
| passed | passed | passed | passed | passed | passed | passed | passed | ||||
11 |
failed
|
failed
|
check
| passed |
⁄
| passed |
check
|
check
| passed | passed | passed | passed | ||
12 |
failed
|
failed
|
failed
|
check
| passed |
⁄
| passed |
check
| passed | passed | passed | passed | ||
13 |
check
|
check
| passed | passed | passed | passed | passed | passed | passed | passed | ||||
14 |
check
| passed | passed | passed |
⁄
| passed | passed | passed | passed | passed | passed | |||
15 |
check
|
check
| passed | passed | passed | passed | passed | passed | passed | passed | ||||
16 | passed | passed |
check
|
check
|
⁄
| passed | passed | passed | passed | passed | passed | passed | ||
17 |
failed
|
failed
| passed | passed | passed |
check
| passed | passed | passed | passed | passed | |||
18 | passed |
failed
| passed | passed |
⁄
| passed | passed | passed | passed | passed | ||||
19 |
check
|
failed
|
failed
| passed |
⁄
|
check
| passed |
check
| passed | passed |
failed
|
check
| ||
20 | passed |
check
| passed | passed | passed |
failed
| passed | passed | passed | passed | passed | |||
21 |
check
|
check
|
failed
|
check
|
⁄
|
failed
|
failed
| passed | passed |
check
|
failed
| passed | ||
22 | passed |
check
|
check
|
check
| passed |
failed
| passed | passed | passed | passed | passed | |||
23 | passed |
check
| passed | passed | passed | passed | passed | passed | passed | passed | ||||
24 | passed | passed | passed | passed | passed | passed | passed | passed | passed | passed | ||||
25 | passed |
failed
| passed | passed |
⁄
| passed | passed | passed | passed | passed | passed | |||
26 |
check
|
check
| passed | passed | passed |
check
| passed | passed | passed | passed |
check
|
check
| ||
27 |
check
| passed | passed | passed | passed | passed |
failed
|
⁄
|
⁄
|
⁄
| ||||
28 | passed | passed | passed | passed | passed |
failed
|
failed
| passed | passed | passed | passed | |||
29 |
check
|
check
| passed | passed | passed | passed | passed | passed | passed | passed | ||||
30 | passed | passed | passed | passed |
failed
|
check
|
check
| passed | passed | passed | passed | passed | ||
31 |
failed
|
failed
| passed | passed |
⁄
| passed | passed | passed | passed | passed | ||||
32 | passed | passed | passed | passed | passed | passed | passed | passed | passed | passed | ||||
33 | passed | passed |
check
|
check
| passed |
⁄
|
check
| passed | passed | passed | passed | passed | ||
34 |
check
|
check
| passed | passed |
⁄
|
failed
| passed | passed | passed | passed | passed | |||
35 |
check
|
failed
| passed | passed |
⁄
|
failed
| passed | passed | passed | passed | passed | |||
36 | passed | passed | passed | passed |
failed
|
failed
| passed | passed | passed | passed | passed | |||
37 |
check
| passed |
check
|
failed
| passed | passed |
failed
|
failed
| passed | passed | passed |
check
|
check
| |
38 | passed | passed |
check
|
check
| passed | passed | passed | passed | passed | passed |
failed
|
failed
| ||
39 |
check
|
check
| passed |
check
| passed |
check
| passed | passed | passed | passed | passed | |||
40 |
check
|
check
| passed | passed |
⁄
|
failed
| passed | passed | passed | passed | passed |
Comparison of the controls of conversion obtained for the three markers on the same DNA sample
Quantitative analysis of methylation levels for cryo-preserved and FFPE tissues (DNA extracted with the dedicated kit)
Identity | Levels of LINE-1methylation (% of methylation) | Difference of methylation | Levels of MLH1methylation (% of methylation) | Difference of methylation | Levels of MGMTmethylation (% of methylation) | Difference of methylation | |||
---|---|---|---|---|---|---|---|---|---|
DNA from cryo-preserved tissues | DNA from FFPE tissues | DNA from cryo-preserved tissues | DNA from FFPE tissues | DNA from cryo-preserved tissues | DNA from FFPE tissues | ||||
1 | 62.0 | 66.3 | -4.3 | 51.5 | 57.7 |
-6.2
| 1.7 | 4.3 | -2.6 |
2 | 66.1 | 67.2 | -1.1 | 4.8 | - |
-
| 2.0 | 6.0 | -4.0 |
3 | 64.6 | 64.8 † | -0.2 | 47.6 | - |
-
| 57.5 | 71.9 ‡ |
-14.3
|
4 | 57.2 | 52.3 | 5.0 | 6.8 | - |
-
| 55.8 | 49.3 |
6.5
|
5 | 61.6 | 64.2 † | -2.6 | 5.3 | 1,7* | 3.6 | 1.9 | 6.2 | -4.2 |
6 | 47.5 | 49.2 | -1.7 | 2.9 | 3.9 | -1.0 | 38.6 | 46.1 |
-7.5
|
7 | 59.4 | 56.6 † | 2.9 | 5.1 | 1.9 | 3.3 | 59.4 † | 43.4 |
16.0
|
8 | 47.9 | 46.5 | 1.3 | 3.4 | 1,5* | 1.9 | 1.7 | 5.7 | -4.0 |
9 | 64.5 | 61.2 | 3.3 | 33.1 | 13.3 |
19.7
| 14.7 | 18.2 | -3.5 |
10 | 61.4 | 54.1 |
7.3
| 5.6 | 1.4 | 4.2 | 19.2 | 32.3 |
-13.1
|
11 | 60.5 | 50.1 |
10.4
| 3.9 | 3,4* | 0.6 | 19.8 | 35.6 † |
-15.8
|
12 | 60.2 | 62.2 † | -2.0 | 4.1 | 2.8 | 1.3 | 2.0 | 5.1 | -3.2 |
13 | 45.1 | 62.5 |
-17.5
| 45.2 | 2.6 |
42.7
| 2.1 | 4.3 | -2.2 |
14 | 57.0 | 60.8 | -3.8 | 3.0 | 7,1* | -4.1 | 1.4 | 1.9 | -0.5 |
15 | 59.2 | 63.2 | -4.0 | 2.5 | 5.6 | -3.1 | 1.4 | 4.9 | -3.5 |
16 | 52.8 | 47.5 † | 5.2 | 2.6 | 3,3* | -0.7 | 1.6 | 5.9 | -4.3 |
17 | 61.0 | 55.6 | 5.3 | 77.7 | 4.6 |
73.1
| 1.7 | 30.2 |
-28.5
|
18 | 59.6 | 53.5 |
6.2
| 4.8 | - |
-
| 28.9 | 25.3 | 3.6 |
19 | 43.3 | 54.4 |
-11.2
| 2.2 |
-
| 73.9 † | 62.2 |
11.7
| |
20 | 61.7 | 57.6 | 4.1 | 4.6 | 3.5 | 1.1 | 15.7 | 25.2 |
-9.5
|
21 | 65.2 | 66.1 † | -1.0 | 6.1 † | - | - | 2.2 | 7.0 ‡ | -4.8 |
22 | 50.9 | 49.1 † | 1.8 | 3.5 | 3.3 | 0.3 | 1.9 | 6.8 | -5.0 |
23 | 61.6 | 59.4 | 2.2 | 3.5 | 2.1 | 1.4 | 1.6 | 4.6 | -2.9 |
24 | 59.3 | 58.0 | 1.4 | 2.4 | 3.5 | -1.1 | 20.8 | 32.4 |
-11.7
|
25 | 61.7 | 71.0 |
-9.3
| 4.1 | - |
-
| 31.7 | 1,9* |
29.8
|
26 | 56.7 | 52.2 | 4.4 | 2.4 | 0.9 | 1.5 | 52.8 † | 20.4 |
32.4
|
27 | 45.5 ‡ | 44.3 | 1.3 | - | 5.9 | - | - | 4.2 |
-
|
28 | 65.3 | 57.4 |
7.9
| 6.6 | 2,7* | 3.9 | 41.8 | 25.2 ‡ |
16.6
|
29 | 64.0 | 57.3 |
6.7
| 3.4 | 2,4* | 1.0 | 2.2 | 3.3 | -1.1 |
30 | 56.4 | 56.7 | -0.3 | 11.5 | 7.3 | 4.3 | 3.9 | 5.3 | -1.5 |
31 | 62.0 | 62.9 | -1.0 | 32.8 | - | - | 5.7 | 5.3 | 0.4 |
32 | 67.2 | 62.0 | 5.2 | 3.9 | 5.2 | -1.2 | 19.0 | 51.8 |
-32.8
|
33 | 60.2 | 62.4 † | -2.3 | 4.0 | 3.9 | 0.1 | 2.0 | 7.9 | -5.9 |
34 | 62.0 | 61.7 | 0.3 | 3.6 | - | - | 2.2 | 5.9 | -3.7 |
35 | 56.9 | 53.3 | 3.5 | 3.8 | - | - | 2.1 | 5.6 | -3.5 |
36 | 40.5 | 41.1 | -0.6 | 3.0 | 3.6 | -0.6 | 1.7 | 5.2 | -3.5 |
37 | 59.0 | 62.1 † | -3.0 | 2.7 | 3.3 | -0.6 | 88.3 † | 73.1 ‡ |
15.1
|
38 | 62.3 | 58.5 † | 3.8 | 4.3 | 1.5 | 2.8 | 68.2 | 71.7 | -3.5 |
39 | 50.4 | 53.2 | -2.9 | 4.8 | 4.5 | 0.3 | 1.9 | 4.6 | -2.7 |
40 | 36.2 | 34.7 | 1.5 | 3.1 | - |
-
| 2.0 | 5.9 | -4.0 |
Synthetic analysis of bisulfite conversion quality and methylation levels for cryo-preserved and FFPE tissues for the three markers (Table 4)
LINE-1
| 1 | 2 | 4 | 6 |
8
| 9 |
14
|
15
| 17 | 20 |
23
| 24 | 26 | 30 | 31 | 32 | 34 | 35 | 36 |
39
| 40 | ||||||||||||||
MLH1
| 5 | 6 | 7 |
8
| 10 | 11 | 12 |
14
|
15
| 16 | 20 | 22 |
23
| 24 | 26 | 28 | 29 | 32 | 33 | 37 | 38 |
39
| |||||||||||||
MGMT
| 1 | 2 | 5 |
8
| 9 | 12 | 13 |
14
|
15
| 16 | 18 | 22 |
23
| 29 | 30 | 31 | 33 | 34 | 35 | 36 | 38 |
39
| 40 |