NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1327643 Query DataSets for GSM1327643
Status Public on Mar 11, 2014
Title ESCC-083T
Sample type genomic
 
Channel 1
Source name tumor DNA from esophageal
Organism Homo sapiens
Characteristics gender: F
age: 70
survival (days): 1422
tnm: T3N1M0
tissue type: esophageal tumor
Extracted molecule genomic DNA
Extraction protocol Use QIAamp DNA Blood Mini kit to extract genomic DNA. 1. Pipet 20 μl QIAGEN Protease (or proteinase K) into the bottom of a 1.5 ml microcentrifuge tube. 2. Add 200 μl whole blood in 200 μl PBS. Add 4 μl of an RNase A stock solution (100 mg/ml) to the sample. 3. Add 200 μl Buffer AL to the sample. Mix by pulse-vortexing for 15 s. 4. Incubate at 56°C for 10 min. DNA yield reaches a maximum after lysis for 10 min at 56°C. 5. Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid. 6. Add 200 μl ethanol (96–100%) to the sample, and mix again by pulse-vortexing for 15 s. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid. 7. Carefully apply the mixture from step 6 to the QIAamp Mini spin column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate. 8. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW1 without wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the collection tube containing the filtrate. 9. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. 10. Recommended: Place the QIAamp Mini spin column in a new 2 ml collection tube and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min. 11. Place the QIAamp Mini spin column in a clean 1.5 ml microcentrifuge tube, and discard the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and add 200 μl Buffer AE or distilled water. Incubate at room temperature (15–25°C) for 1 min, and then centrifuge at 6000 x g (8000 rpm) for 1 min.
Label Cy3
Label protocol 1. Add 4μL β-mercaptoethanol to 2200μL Random Primer Buffer, Mix by pulse-vortexing. Use 1050 μL Random Primer Buffer (containing β-mercaptoethanol) to dilute cy3-Random Nonamers and cy5-Random Nonamers respectively. 2. Add 500ng genomic DNA to LHC(Labeling and Hybridization Control). Then add 2μLLCH-1 to test Sample, and 2μLLCH-2 to Reference Sample, amount to 40μL respectively. Add test Sample (containing LLCH-1) to cy3-Random Nonamers, and Reference Sample (containing LLCH-2) to cy5-Random Nonamers, amount to 80μL respectively. 98 °C for 10 min，4°C for 2min. 3. Prepare dNTP/Klenow Master Mix (20μL): 8μL Nuclease-free Water, 10μL 10mM dNTP Mix, 2μL Klenow Fragment (3’->5’ exo-)50U/μl. 4. Add dNTP/Klenow Master Mix (20μL) to Sample mixtures (80μL) respectively. 37 °C for 2 hr. 5. Add 21.5μL Stop Solution to stop reaction. 6. Then purify the label reaction liquid.
 
Channel 2
Source name normal DNA from blood
Organism Homo sapiens
Characteristics gender: F
age: 70
survival (days): 1422
tnm: T3N1M0
tissue type: normal blood
Extracted molecule genomic DNA
Extraction protocol Use QIAamp DNA Blood Mini kit to extract genomic DNA. 1. Pipet 20 μl QIAGEN Protease (or proteinase K) into the bottom of a 1.5 ml microcentrifuge tube. 2. Add 200 μl whole blood in 200 μl PBS. Add 4 μl of an RNase A stock solution (100 mg/ml) to the sample. 3. Add 200 μl Buffer AL to the sample. Mix by pulse-vortexing for 15 s. 4. Incubate at 56°C for 10 min. DNA yield reaches a maximum after lysis for 10 min at 56°C. 5. Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid. 6. Add 200 μl ethanol (96–100%) to the sample, and mix again by pulse-vortexing for 15 s. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid. 7. Carefully apply the mixture from step 6 to the QIAamp Mini spin column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate. 8. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW1 without wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the collection tube containing the filtrate. 9. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. 10. Recommended: Place the QIAamp Mini spin column in a new 2 ml collection tube and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min. 11. Place the QIAamp Mini spin column in a clean 1.5 ml microcentrifuge tube, and discard the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and add 200 μl Buffer AE or distilled water. Incubate at room temperature (15–25°C) for 1 min, and then centrifuge at 6000 x g (8000 rpm) for 1 min.
Label Cy5
Label protocol 1. Add 4μL β-mercaptoethanol to 2200μL Random Primer Buffer, Mix by pulse-vortexing. Use 1050 μL Random Primer Buffer (containing β-mercaptoethanol) to dilute cy3-Random Nonamers and cy5-Random Nonamers respectively. 2. Add 500ng genomic DNA to LHC(Labeling and Hybridization Control). Then add 2μLLCH-1 to test Sample, and 2μLLCH-2 to Reference Sample, amount to 40μL respectively. Add test Sample (containing LLCH-1) to cy3-Random Nonamers, and Reference Sample (containing LLCH-2) to cy5-Random Nonamers, amount to 80μL respectively. 98 °C for 10 min，4°C for 2min. 3. Prepare dNTP/Klenow Master Mix (20μL): 8μL Nuclease-free Water, 10μL 10mM dNTP Mix, 2μL Klenow Fragment (3’->5’ exo-)50U/μl. 4. Add dNTP/Klenow Master Mix (20μL) to Sample mixtures (80μL) respectively. 37 °C for 2 hr. 5. Add 21.5μL Stop Solution to stop reaction. 6. Then purify the label reaction liquid.
 
 
Hybridization protocol The labeled genomic DNA (31μg) was dried at 45℃ and diluted in 5.6 μl Sample Tracking Control. Then hybridized in 14.4 μl Hybridization solution (containing 2X Hybridization Buffer, Hybridization Component A, Alignment Oligo). Arrays were hybridized in Maui hybridization stations for 40-72 hr at 42 °C, and then washed in 42 °C with buffer (containing VWR Water, 10X Wash Buffer (I, II or III), 1M DTT) by using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html).
Scan protocol Arrays were scanned on a NimbleGen MS 200 Microarray Scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Description Esophageal Cancer Project
Data processing Arrays were processed using NimbleScan 2.6 software,which transformed image signal to digital signal, following using segMNT algorithm to analyse the normalized signal ratio.
Final vaues are reported as 'log2 (EXP_NORM/REF_NORM)' in linked segMNT supplementary files.
 
Submission date Feb 13, 2014
Last update date Mar 12, 2014
Contact name Lin Li
E-mail(s) lilin@genomics.cn
Organization name BGI
Street address Beishan Industrial Zone
City shenzhen
ZIP/Postal code 518083
Country China
 
Platform ID GPL15436
Series (2)
GSE54994 a-CGH of Esophageal Cancer (53 cases)
GSE54995 Esophageal Cancer Project

Supplementary file Size Download File type/resource
GSM1327643_539725A03_2012-07-19_532.pair.gz 13.8 Mb (ftp)(http) PAIR
GSM1327643_539725A03_2012-07-19_635.pair.gz 13.8 Mb (ftp)(http) PAIR
GSM1327643_539725A03_2012-07-19_segMNT.txt.gz 27.8 Mb (ftp)(http) TXT
Processed data provided as supplementary file
| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap