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Status |
Public on Mar 09, 2010 |
Title |
HONE1 cell line replicate 2 |
Sample type |
genomic |
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Channel 1 |
Source name |
epithelial biopsies of carcinoma
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Organism |
Homo sapiens |
Characteristics |
tissue: nasopharynx cell line: HONE1 disease state: carcinoma
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Treatment protocol |
Both cell lines were without any treatment before extraction of genomic DNA.
|
Growth protocol |
The TW01 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). The HONE1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA were isolated by using DNeasy Tissue Kit (Qiagen) according to the manufacturer's instructions.
|
Label |
Cy5
|
Label protocol |
Genomic DNA was completely fragmented with AluI and RsaI (Promega, Madison, WI, USA ), followed by purification with QIAprep Spin Miniprep Kit (Qiagen). All of the digested DNAs were subjected to labeling reactions using Agilent’s Genomic DNA Labeling Kit PLUS per manufacturer’s instructions. For each array-CGH experiment, we used DNA isolated from NPC cell line as experimental genome (cyanine 5 UTP-labeled) and the commercial healthy Caucasian male genome (Promega) as reference (cyanine 3 UTP-labeled).
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Channel 2 |
Source name |
peripheral lymphocytes
|
Organism |
Homo sapiens |
Characteristics |
disease state: normal male reference
|
Treatment protocol |
Both cell lines were without any treatment before extraction of genomic DNA.
|
Growth protocol |
The TW01 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). The HONE1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA were isolated by using DNeasy Tissue Kit (Qiagen) according to the manufacturer's instructions.
|
Label |
Cy3
|
Label protocol |
Genomic DNA was completely fragmented with AluI and RsaI (Promega, Madison, WI, USA ), followed by purification with QIAprep Spin Miniprep Kit (Qiagen). All of the digested DNAs were subjected to labeling reactions using Agilent’s Genomic DNA Labeling Kit PLUS per manufacturer’s instructions. For each array-CGH experiment, we used DNA isolated from NPC cell line as experimental genome (cyanine 5 UTP-labeled) and the commercial healthy Caucasian male genome (Promega) as reference (cyanine 3 UTP-labeled).
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Hybridization protocol |
Array hybridizations were carried out with 490μl of hybridization mixtures 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).
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Scan protocol |
The hybridized arrays were scanned using an Agilent G2565BA DNA microarray scanner (Agilent Technologies). The ScanControl Software (Agilent Technologies) was used for image acquisition. In brief, the arrays were imaged at a resolution of 5 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 array 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 |
Biological replicate 2 of 2. genomic DNA of NPC cell lines vs. pool of normal male genome .
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Data processing |
After image acquisition, data extraction, normalization and transformation were carried out by using Agilent Feature Extraction Software, version 8.1.1 (Agilent Technologies), procedures listed as follow: (1) Place Grid, find and measure spots -define what is the array format of the image to be processed. (2) Flag outliers a. Compute population outliers- flag features and local backgrounds as population outliers if the minimum number of replicates is met and the mean signals varies significantly from the distribution. b. Compute NonUniform outliers- flag features and local backgrounds as NonUniform if the pixel distribution is outside the boundary defined by the three-parameter noise model. (3) Compute Background, bias and error Background subtraction→ signal correction → choose error model (4) Dye normalization – a. Use rank consistent probes. b. Normalization correction methods: Linear method (5) Compute ratios- the log ratio values are capped to 4 with appropriate signs in positive and negative directions. Subsequent data analysis was performed with the other custom analytical software, CGH Analytics (version 3.5.14, Agilent Technologies). The Linear smoothing algorithm is used in the Moving Average approach to smooth the data sets with window size of 5 Mb.
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Submission date |
Mar 10, 2009 |
Last update date |
Mar 09, 2010 |
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 |
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Platform ID |
GPL4091 |
Series (2) |
GSE15172 |
Copy number variation analysis of nasopharyngeal carcinoma cell lines |
GSE15191 |
Nasopharyngeal carcinoma cell lines TW01 and HONE1: transcriptomic and genomic analyses |
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