Background
Methods
Samples processing and DNA extraction
RAPD-PCR analysis and scoring of alterations
RAPD Primers | |||||
---|---|---|---|---|---|
Serial no. | RAPD Primer ID | Sequence (5'-3') | GC content (%age) | Annealing temp. | MgCl2 (mM) |
1. |
80/07
|
GCACGCCGGA
| 80 | 38°C | 3.0 |
2. |
80/01
|
GCACCCGACG
| 80 | 35°C | 3.0 |
3. |
70/04
|
CGCATTCCGC
| 80 | 35°C | 3.0 |
4. |
70/01
|
CATCCCGAAC
| 70 | 38°C | 3.0 |
5. |
60/31
|
TGCGCGATCG
| 70 | 38°C | 3.0 |
Microsatellite markers
| |||||
Serial no. |
Microsatellite marker ID
|
Primer sequences (5'-3')
|
Annealing temp. |
Product size (bp)
| |
1. |
D4S3173
|
Left Primer: TTGGAGTCCCTGAAGGACC
Right primer: TACAAACACCAAGCCCCTTC
| 57°C | 103–104 | |
2. |
D4S2827
|
Left Primer: GACGGGAGAGAAAATGCATT
Right primer: TCAATAAGCACCAAAACTACTCAG
| 55°C | 132–133 | |
3. |
D4S1295
|
Left Primer:TAACAAAACCATCTCCAAACTTCTC
Right primer: GTGGACAGTACAACACAGTTGAC
| 57°C | 170 | |
4. |
D4S2643
|
Left Primer:TTGTCCAATAACTCTTTTCCTAACA
Right primer: CTGATTCCTATATCAATCTTGGCC
| 57°C | 179–180 | |
5. |
D4S2672
|
Left Primer: TAAAGATGTGATTTGTATTGCATTG
Right primer: AGTTTCAGCCTCTCAAAATTTCA
| 57°C | 199–200 | |
SNP markers
| |||||
Serial no. |
SNP Cluster ID
|
Primer sequences (5'-3')
|
Annealing temp. |
Product size (bp)
|
Restriction endonucleases
|
1. |
rs2276930
| Left primer: GCCGTAACTAACCTCGGCATC Right primer: GCCTCACGCTCCCCGAGCGCA | 61°C | 270 | BstN1 |
2. |
rs3733414
| Left primer: CGCCCCAGTTCTCTTCTGT Right primer: TGTTGTTACTTCAAGTTCAAAATGG | 60.8°C | 290 | Rsa1 |
3. |
rs455600
| Left primer: TCATTGACTTTTGCTTTTCCTAA Right primer: CCAACGAATCAGCAAAGAAC | 60.8°C | 299 | Acc1 |
4. |
rs213090
| Left primer: GGCAGTTGAAATTTACTTTATAGTTCT Right primer: AGACTGCAGTGTGCAATTCTG | 61°C | 394 | HaeIII |
5. |
rs450320
| Left primer: CGAAATCCTACTCCTGGCTTT Right primer: GCATCACTCCTGCTCCTCAT | 60.8°C | 349 | Hpy1881 |
6. |
rs7663350
| Left primer: TGGTTTTGGTGAAGCATAAGAA Right primer: AAGTTATCCAGGATTTCAATCTCA | 60.8°C | 400 | Sau3A1 |
7. |
rs1193110
| Left primer: AGACCCTTTCAGCCAGTTCA Right primer: AGTGAGATGAGGCCAGTGCT | 61°C | 369 | Msp1 |
8. |
rs1298865
| Left primer: TTGTCATCCTTCCTTTTAGCAA Right primer: GATCCTTCAATGAATATGTTCTTCTC | 60.8°C | 400 | Dde1 |
Characterization of altered fragment
Characterization of the RAPD primer binding sites
Loss of heterozygosity (LOH) studies
Microsatellite Markers: Selection and analysis
SNP markers: Selection and analysis
RNA extraction and Reverse transcription- PCR analysis
Results
RAPD-fingerprinting of human astrocytic tumors and characterization
Characterization of the RAPD primer binding sites
LOH analysis of the FATlocus
Microsatellite markers D4S | D4S3173 | D4S2827 | D4S1295 | D4S2643 | D4S2672 | |
---|---|---|---|---|---|---|
Informative samples
|
Grade II
| U | U | 6/20 (30%) | U | U |
Grade IV
| U | U | 5/20 (25%) | U | U | |
LOH per informative samples
|
Grade II
| U | U | 3/6 (50%) | U | U |
Grade IV
| U | U | 2/5 (40%) | U | U | |
Total LOH per informative samples
(Grade II & IV)
| U | U |
5/11 (45.45%)
| U | U |
Samples
Low grade Diffuse Astrocytoma (Grade II) | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
SNP markers | A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 | A9 | A10 | A11 | A12 | A13 | A14 | A15 | A16 | A17 | A18 | A19 | A20 |
rs2276930
| U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U |
rs3733414
|
NL
|
NL
| U | U | U |
L
|
L
|
NL
|
NL
|
NL
|
NL
|
NL
|
NL
|
NL
|
NL
|
NL
| U |
NL
|
NL
| U |
rs455600
| U | U | U | U | U |
L
| U |
NL
|
NL
| U |
NL
| U |
NL
| U | U | U |
L
|
NL
| U | U |
rs213090
|
NL
|
NL
| U | U |
NL
| U |
L
| U |
NL
| U |
NL
| U | U | U | U | U |
L
|
NL
|
L
| U |
rs450320
| U |
L
| U |
L
|
NL
| U |
NL
| U |
L
|
NL
| U |
NL
| U |
NL
|
NL
|
NL
| U |
NL
| U |
L
|
rs7663350
|
NL
|
NL
| U | U |
NL
|
NL
| U | U | U | U | U |
NL
| U | U | U |
NL
| U | U | U |
NL
|
rs1193110
| U |
NL
| U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U |
NL
| U |
rs1298865
| U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U |
Glioblastoma multiforme (Grade IV)
| ||||||||||||||||||||
SNP markers
|
G1
|
G2
|
G3
|
G4
|
G5
|
G6
|
G7
|
G8
|
G9
|
G10
|
G11
|
G12
|
G13
|
G14
|
G15
|
G16
|
G17
|
G18
|
G19
|
G20
|
rs2276930
| U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U | U |
rs3733414
| U | U | U | U |
NL
| U |
L
| U | U |
L
|
L
| U | U |
NL
|
NL
| U | U | U |
NL
|
NL
|
rs455600
|
NL
| U |
NL
| U | U | U |
L
| U |
L
| U |
NA
|
NL
| U | U | U | U | U | U | U |
NL
|
rs213090
|
L
|
L
| U | U | U | U | U |
NL
| U | U |
NL
| U |
NL
| U | U | U | U | U | U |
L
|
rs450320
|
NL
|
NL
|
L
|
L
| U |
NL
| U |
L
|
NA
|
NL
|
L
| U | U |
NL
| U | U |
L
| U | U | U |
rs7663350
| U | U |
NL
| U | U |
NL
| U |
U
|
NL
| U | U | U | U | U | U | U | U |
NL
|
NL
|
NL
|
rs1193110
| U | U | U | U | U | U | U | U | U |
L
| U |
NL
| U | U | U | U | U | U | U | U |
rs1298865
| U | U | U | U | U | U | U | U | U | U | U |
L
| U | U | U | U | U | U | U | U |
Summary of LOH detected with each SNP markers | |||||||||
---|---|---|---|---|---|---|---|---|---|
SNP-Markers
| rs22 76930 | rs37 33414 | rs45 5600 | rs21 3090 | rs45 0320 | rs76 63350 | rs11 93110 | rs12 98865 | |
Informative samples
|
Grade II
| 0/20 (0%) | 15/20 (75%) | 7/20 (35%) | 9/20 (45%) | 12/20 (60%) | 7/20 (35%) | 2/20 (10%) | 0/20 (0%) |
Grade IV
| 0/20 (0%) | 8/20 (40%) | 6/20 (30%) | 6/20 (30%) | 10/20 (50%) | 0/20 (0%) | 2/20 (10%) | 1/20 (5%) | |
LOH per informative samples
|
Grade II
| 0/20 (0%) | 2/15 (13.33%) | 2/7 (28.57%) | 3/9 (33.33%) | 4/12 (33.33%) | 0/7 (0%) | 0/2 (0%) | 0/0 (0%) |
Grade IV
| 0/20 (0%) | 3/8 (37.5%) | 2/6 (33.33%) | 3/6 (50%) | 5/10 (50%) | 0/0 (0%) | 1/2 (50%) | 1/1 (100%) | |
Total LOH
(Grade II & IV)
|
0/20 (0%)
|
5/23
21.73%
|
4/13
30.73%
|
6/15
40%
|
9/22
40.90%
|
0/7
(0%)
|
1/4
25%
|
1/1 100%
| |
LOH per informative samples
| |||||||||
Samples
|
Grade II (N = 20)
|
Grade IV (N = 20)
|
Grade II&IV (N = 40)
| ||||||
Overall SNP LOH per
informative samples
|
8/19*
(42.10%)
|
11/19*
(57.89%)
|
19/38
(50%)
|