|
| Status |
Public on Nov 01, 2020 |
| Title |
Urinary Bladder Cancer Tissue #S51 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Urinary Bladder Carcinoma tissue
|
| Organism |
Homo sapiens |
| Characteristics |
age: Age: 65
Stage: Stage T1
grade: Grade High
smoking history: NO
alcohol ingestion: NO
drinking water source: underground
gender: M
arsenic in tumor tissue: 162.35ppb
occupation of the subject: fish business
|
| Treatment protocol |
tumor tissues vs normal peripheral blood lymphocytes
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA isolation from peripheral blood lymphocytes (N) and cancer tissues (T), using standrad Phenol/ Chloroform method. Tumor tissue were microdissected for tumor cell enrichment before DNA extraction.
|
| Label |
Cy5
|
| Label protocol |
Agilent Direct method has been used for the sample processing. About 1000 ng of control and test DNA was used for the restriction digestion in the master mix containing AluI and RsaI restriction enzymes as per manufactures recommendation. The samples were incubated at 37°C for 2 hours followed by heat inactivation of enzymes at 65°C for 20 minutes. To confirm the efficiency of restriction enzymes to obtain fragments of size 200-500bp, about 2 μL of the digested gDNA was tested on a 0.8% agarose gel. Labeling of samples was done by random priming method, in which the random hexamers, Cy3-dUTP & Cy5-dUTP, dNTP, Buffer and Klenow enzyme was used. Briefly, 1X Random primer mix was added to each of 26µl digested control and test samples. The DNA was denatured at 95 °C for 3 minutes followed by snap chill on ice for 5 minutes. Master mix for Cy3 and Cy5 dNTPs was done separately to ensure that, the control sample is labeled with Cy3 and test sample with Cy5 respectively. About 19 ul of labeling master mix prepared as per manufacturers recommendation was added to the denatured control and test DNA sample and incubated at 37°C for 2 hours followed by enzyme heat inactivation at 65°C for 10 minutes. The labeled samples were cleaned up by Amicon 30kDa filter size exclusion filter.
|
| |
|
| Channel 2 |
| Source name |
Peripheral Blood lymphocytes
|
| Organism |
Homo sapiens |
| Characteristics |
gender: M
occupation of the subject: fish business
reference: matched normal blood
|
| Treatment protocol |
tumor tissues vs normal peripheral blood lymphocytes
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA isolation from peripheral blood lymphocytes (N) and cancer tissues (T), using standrad Phenol/ Chloroform method. Tumor tissue were microdissected for tumor cell enrichment before DNA extraction.
|
| Label |
Cy3
|
| Label protocol |
Agilent Direct method has been used for the sample processing. About 1000 ng of control and test DNA was used for the restriction digestion in the master mix containing AluI and RsaI restriction enzymes as per manufactures recommendation. The samples were incubated at 37°C for 2 hours followed by heat inactivation of enzymes at 65°C for 20 minutes. To confirm the efficiency of restriction enzymes to obtain fragments of size 200-500bp, about 2 μL of the digested gDNA was tested on a 0.8% agarose gel. Labeling of samples was done by random priming method, in which the random hexamers, Cy3-dUTP & Cy5-dUTP, dNTP, Buffer and Klenow enzyme was used. Briefly, 1X Random primer mix was added to each of 26µl digested control and test samples. The DNA was denatured at 95 °C for 3 minutes followed by snap chill on ice for 5 minutes. Master mix for Cy3 and Cy5 dNTPs was done separately to ensure that, the control sample is labeled with Cy3 and test sample with Cy5 respectively. About 19 ul of labeling master mix prepared as per manufacturers recommendation was added to the denatured control and test DNA sample and incubated at 37°C for 2 hours followed by enzyme heat inactivation at 65°C for 10 minutes. The labeled samples were cleaned up by Amicon 30kDa filter size exclusion filter.
|
| |
|
| |
| Hybridization protocol |
Equal amount of labeled Test & Control DNA sample was added into a fresh tube containing 25 µl of Human Cot-1 DNA (1mg/ml), 26ul of Agilent 10X blocking agent and 130ul Agilent 2X hybridization buffer. The total hybridization volume was 45ul. The above hybridization mix was denatured at 95°C for 3 minutes and incubated the microfuge tubes at 37°C for 30 minutes. The samples were hybridized at 65°C for 40 hours in the hybridization chamber. After hybridization, the slides were washed using aCGH Wash Buffer1 (Agilent Technologies, Part Number 5188-5221) at room temperature for 5 minutes and aCGH Wash Buffer 2 (Agilent Technologies, Part Number 5188-5222) at 37°C for 1 minutes. The slides were then washed with Acetonitrile for 10 seconds.
|
| Scan protocol |
Agilent Scanner (Agilent Technologies, Part Number G2565CA).
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software. Feature extracted raw data was normalized using Lowes normalization and analyzed Agilent CytoGenomics 2.9 Software.
|
| |
|
| Submission date |
Nov 14, 2018 |
| Last update date |
Nov 02, 2020 |
| Contact name |
CHINMAY KUMAR PANDA |
| Organization name |
CHITTARANJAN NATIONAL CANCER INSTITUTE, INDIA
|
| Department |
ONCOGENE REGULATION UNIT
|
| Street address |
37, SP MUKHERJEE ROAD, KOLKATA
|
| City |
KOLKATA |
| State/province |
WEST BENGAL |
| ZIP/Postal code |
700008 |
| Country |
India |
| |
|
| Platform ID |
GPL11363 |
| Series (1) |
| GSE122514 |
Landscape of genomic alterations in Indian Urinary Bladder cancer patients |
|
