SssI-treated positive control: 4ug of genomic DNA (normal brain) was treated with 16 units of SssI enzyme and 160 nmol S-Adenosylmethione for 6 hours at 37oC twice. Genomiphied negative control: 10ng of genomic DNA (normal brain) was treated using GenomiPhi V2 Amplification Kit (Amersham Biosciences, Piscataway, NJ) according to manufacture's instructions with unmodified dCTP. Methylation-specific restriction enzyme: 750 ng of each genomic sample was digested with 25 units of BfaI (NEB, MA) for 6 hours at 37oC. Digested DNA was then ligated to oligonucleotide linkers consisting of annealed H-12 (5'-TAATCCCTCGGA-3') and H-24 (5'-AGGCAACTGTGCTATCCGAGGGAT - 3') oligonucleotides. Following ligation, the genomic DNA was divided into two equal portions for separate digestion with 20 units of HpaII (methylation-sensitive isoschizomer) or 40 units of MspI (methylation-insensitive isoschizomer) (NEB). Each digest was subjected to PCR amplification using the same H-12/H-24 primers with the following conditions: 72°C for 5 minutes, followed by 16 cycles of 97°C for 1 minutes, 62°C for 30 seconds, 72°C for 3 minutes, followed by 72°C for 10 minutes.
Growth protocol
U87MG cells were maintained in Dulbecco’s modified Eagle’s medium/F12 (Mediatech, Herndon, VA, USA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 mg/ml streptomycin, were grown in a 5% CO2 humidified atmosphere. U87MG cells were obtained from Dr. P. Mischel (University of California, Los Angeles, CA, USA). Normal brain and Glioblastoma specimens has been collected and archived through an IRB approved protocol and obtained from the UCLA Brain Tumor Translational Resource.
Extracted molecule
genomic DNA
Extraction protocol
DNA was extracted using Qiagen Dneasy Blood and Tissue Kit (Qiagen, Valencia, CA)
Label
Cy5
Label protocol
HpaII and MspI digests were labeled with Cyanine 5-dUTP and Cyanine 3-dUTP respectively (Perkin-Elmer, Waltham, MA) using Bioprime Array CGH Genomics Labeling Module (Invitrogen, Carlsbad, CA) according to manufacture's instructions.
SssI-treated positive control: 4ug of genomic DNA (normal brain) was treated with 16 units of SssI enzyme and 160 nmol S-Adenosylmethione for 6 hours at 37oC twice. Genomiphied negative control: 10ng of genomic DNA (normal brain) was treated using GenomiPhi V2 Amplification Kit (Amersham Biosciences, Piscataway, NJ) according to manufacture's instructions with unmodified dCTP. Methylation-specific restriction enzyme: 750 ng of each genomic sample was digested with 25 units of BfaI (NEB, MA) for 6 hours at 37oC. Digested DNA was then ligated to oligonucleotide linkers consisting of annealed H-12 (5'-TAATCCCTCGGA-3') and H-24 (5'-AGGCAACTGTGCTATCCGAGGGAT - 3') oligonucleotides. Following ligation, the genomic DNA was divided into two equal portions for separate digestion with 20 units of HpaII (methylation-sensitive isoschizomer) or 40 units of MspI (methylation-insensitive isoschizomer) (NEB). Each digest was subjected to PCR amplification using the same H-12/H-24 primers with the following conditions: 72°C for 5 minutes, followed by 16 cycles of 97°C for 1 minutes, 62°C for 30 seconds, 72°C for 3 minutes, followed by 72°C for 10 minutes.
Growth protocol
U87MG cells were maintained in Dulbecco’s modified Eagle’s medium/F12 (Mediatech, Herndon, VA, USA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 mg/ml streptomycin, were grown in a 5% CO2 humidified atmosphere. U87MG cells were obtained from Dr. P. Mischel (University of California, Los Angeles, CA, USA). Normal brain and Glioblastoma specimens has been collected and archived through an IRB approved protocol and obtained from the UCLA Brain Tumor Translational Resource.
Extracted molecule
genomic DNA
Extraction protocol
DNA was extracted using Qiagen Dneasy Blood and Tissue Kit (Qiagen, Valencia, CA)
Label
Cy3
Label protocol
HpaII and MspI digests were labeled with Cyanine 5-dUTP and Cyanine 3-dUTP respectively (Perkin-Elmer, Waltham, MA) using Bioprime Array CGH Genomics Labeling Module (Invitrogen, Carlsbad, CA) according to manufacture's instructions.
Hybridization protocol
Cy-3 and Cy-5 labeled amplicons were co-hybridized at 65oC for 40 hours to Agilent CpG Island microarrays according to manufacture's instructions. After hybridization, slides were washed consecutively.
Scan protocol
Scanned by Agilent DNA Microarray Scanner G2505B
Description
Technical replicate 1 was performed on a different day than technical replicate 2.
Data processing
Agilent Feature Extraction 10.5.3 (FE) was used for extraction only. Data analysis was done on unnormalize data from FE output (rProcessedSignal and gProcessSignal). Probes were mapped to corresponding fragment from BfaI digestion. Loess curve was constructed based on insensitive fragments and Loess correction was applied to the the entire arrays.
