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Status |
Public on May 30, 2007 |
Title |
NA_reactivation_1 time_rep2 |
Sample type |
genomic |
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Channel 1 |
Source name |
EBV-positive cell lines derived from nasopharyngeal carcinoma, without EBV reactivation, low-passage-number
|
Organism |
Homo sapiens |
Characteristics |
Cell line: an EBV-positive NPC cell line Gender: male Tissue: Nasopharyngeal carcinoma
|
Treatment protocol |
without and treatment
|
Growth protocol |
NA cells were all maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 400 microgram/ml of G418
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA was isolated with DNeasy Tissue Kit (Qiagen) according to the manufacturer's instructions
|
Label |
Cy5
|
Label protocol |
Genomic DNA labeling was performed according to the standard array-CGH protocol provided by Agilent Technologies. After 3 μg of genomic DNA was completely fragmented and purified, all of the digested DNAs were subjected to labeling reactions with a Bioprime labeling kit (Invitrogen) according to the manufacturer’s instructions. Labeling reactions were performed in a volume of 50 μl with a modified dNTP pool containing 120 μM each of dATP, dGTP, and dCTP; 60μM dTTP; and 60μM Cy5-dUTP (PerkinElmer). After incubation for 2 h at 37 ℃, the reactions were inactivated.
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Channel 2 |
Source name |
EBV-positive cell lines derived from nasopharyngeal carcinoma, EBV reactivation, one time
|
Organism |
Homo sapiens |
Characteristics |
Cell line: an EBV-positive NPC cell line Gender: male Tissue: Nasopharyngeal carcinoma
|
Treatment protocol |
Induction of EBV reactivation was established by combinational use of 12-O-tetradecanoylphorbol -13-acetate (TPA) and sodium n-butyrate (SB). Briefly, NA cells seeded in cultured medium and allow adhering overnight before treatment. TPA and SB were dissolved in medium at final concentration of 20 ng/ml and 3 mM, respectively. Twenty four hours post incubation, medium containing TPA and SB was removed following by incubation in normal medium for 24 hours prior genomic DNA extraction.
|
Growth protocol |
NA cells were all maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 400 microgran/ml of G418
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA was isolated with DNeasy Tissue Kit (Qiagen) according to the manufacturer's instructions
|
Label |
Cy3
|
Label protocol |
Genomic DNA labeling was performed according to the standard array-CGH protocol provided by Agilent Technologies. After 3 μg of genomic DNA was completely fragmented and purified, all of the digested DNAs were subjected to labeling reactions with a Bioprime labeling kit (Invitrogen) according to the manufacturer’s instructions. Labeling reactions were performed in a volume of 50 μl with a modified dNTP pool containing 120 μM each of dATP, dGTP, and dCTP; 60μM dTTP; and 60μM Cy3-dUTP (PerkinElmer). After incubation for 2 h at 37 ℃, the reactions were inactivated.
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Hybridization protocol |
To prepare the hybridization mixture, the experimental and reference samples were subsequently pooled, purified and concentrated with a Centricon YM-30 column (Millipore), resuspended to a final volume of 150 μl, and then mixed with 50 μg of human Cot-1 DNA (Invitrogen), 50 μl of 10× blocking buffer (Agilent), and 250 μl of 2× aCGH hybrixization buffer (Agilent). Before hybridization, the 500 μl hybridization mixtures were denatured at 95℃ for 3 min and incubated at 37℃ for 30 min. Array hybridizations were carried out for 40 h at 65℃, then washed for 5 min at room temperature in Oligo aCGH Wash Buffer 1 (Agilent), followed by 1 min at 37℃ in Oligo aCGH Wash Buffer 2 (Agilent).
|
Scan protocol |
Hardware: Agilent G2565BA DNA microarray scanner (Agilent Technologies) Software: ScanControl Software (Agilent Technologies) The scanning and image acquisition protocols: the arrays were imaged at a resolution of 10 μm. Excitation of Cy3 and Cy5 was performed at a wavelength of 532 and 635 nm, respectively. A laser power of 100% was used. Each arrays was subjected to a series of scans. The signals were digitized into 16 bit/pixel, yielding a maximum detection range from 1 to 65536; i.e., almost five orders of magnitude.
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Description |
NA is an EBV-positive NPC cell line previously generated by in vitro infection of NPC-TW01 cells with recombinant Akata EBV.
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Data processing |
The data in VALUE column were generated by using Linear Normalization Method with rank consistent probes. The data was transformed into Log10-based scale.
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Submission date |
Nov 30, 2006 |
Last update date |
Mar 23, 2007 |
Contact name |
Chia Huei Lee |
E-mail(s) |
chlee124@nhri.edu.tw
|
Organization name |
Taiwan National Health Research Institute
|
Department |
National Institute of Cancer Research
|
Street address |
35, Keyan Rd.
|
City |
Zhunan Miaoli County |
ZIP/Postal code |
114 |
Country |
Taiwan |
|
|
Platform ID |
GPL2879 |
Series (1) |
GSE6472 |
The influence of highly recurrent EBV reactivation on genetic copy number alterations |
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