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| Status |
Public on Mar 31, 2019 |
| Title |
Tumor breast tissue_patient with obesity_non-hereditary breast cancer (Patient 5) |
| Sample type |
genomic |
| |
|
| Source name |
Tumor breast tissue from a patient with obesity diagnosed with non-hereditary breast cancer (Patient 5)
|
| Organism |
Homo sapiens |
| Characteristics |
individual: Patient 5
tissue: Breast tumor tissue
bmi: Obese BMI
disease state: non-hereditary breast cancer
|
| Treatment protocol |
The samples were obtained through the Instituto FUCAM AC. All patients signed informed consent letter. The sample of tumor tissue was used both for the diagnosis of breast cancer and for this research protocol.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA of all patients was isolated from blood leukocytes using the salting out procedure described by Miller et al., (17); DNA from tumor tissue samples was obtained from surgical specimens, using the DNeasy Blood & Tissue Kit (QIAGEN GmbH, Hilden, Germany), following those conditions recommended by the manufacturer.
|
| Label |
Biotin
|
| Label protocol |
The entire mitochondrial DNA sequence was amplified in three overlapping long PCR fragments, with each reaction containing 50 ng of genomic DNA. The primers for PCR amplification were the same as in the previous report for MitoChip V2.1. Affymetrix fragmentation reagent (0.2 U of DNase I/g DNA), 5 l of OnePhorAll buffer (Amersham Life Sciences, Arlington Heights, IL), and EB buffer. Samples were then incubated at 95°C for 15 minutes to inactivate DNase I. Fragmented DNA was labeled by adding 2.0 l of GeneChip DNA labeling reagent and 3.4 l of 30 U/l terminal deoxynucleotidyl transferase (both from Affymetrix).
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| |
|
| Hybridization protocol |
Prehybridization, hybridization, washing, and scanning of the MitoChip were performed as described in the Affymetrix CustomSeq Resequencing protocol. The prehybridizations were performed for 15 minutes in 80-l (for v2.0 chips) solution containing 3 mol/L tetramethylammonium chloride, 0.1% Tween 20, and 10 mmol/L Tris, pH 7.8. The chips were hybridized for 16 hours at 48°C with 60 rpm rotation in a hybridization solution containing 3 mol/L tetramethylammonium chloride, 100 g/ml herring sperm DNA, 500 g/ml bovine serum albumin, 10 mmol/L Tris, pH 7.8, 0.01% Tween 20, and 200 pmol/L control oligo. The chips were then washed on the Affymetrix fluidics station 450 using the program Mini_DNAArray_WS5_450_FELXMini_DNAArray_WS5_450.
|
| Scan protocol |
Chips were scanned in Affymetrix GeneChip Scanner 3000 and data acquisition was performed using the Affymetrix Genechip Command Console (AGCC) software.
|
| Description |
Selective amplication of mtDNA
|
| Data processing |
Analysis of microarray data from the MitoChips v2.0 was carried out using the GeneChip Sequence Analysis Software (GSEQ) v4.0, in order to assign to each position, the basis that meets the quality criteria in the mitochondrial genome. Mitochondrial tumor DNA sequence was compared to matched leukocyte mitochondrial DNA sequence in order to identify true mutations rather than polymorphisms. Data analysis was carried out with GSEQ 4.1 with “model type” set at diploid to enable the detection of heteroplasmy and “quality score threshold” set at 3 to provide the best base calling accuracy and rate. The other parameters used by the algorithm were No Signal Threshold= 2, Weak Signal Fold Threshold= 20, Large SNR Threshold= 12, Min Fraction of Calls of Samples = 0.5, Trace Threshold = 1, Sequence Profile Threshold = -0.175
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| |
|
| Submission date |
Jan 28, 2019 |
| Last update date |
Apr 01, 2019 |
| Contact name |
Nishi Alan Adams-Reyes |
| Phone |
2223411316
|
| Organization name |
Universidad Nacional Autónoma de México
|
| Department |
Faculty of Medicine
|
| Lab |
Obesity Research Unit
|
| Street address |
AV. UNIVERSIDAD 3000
|
| City |
Ciudad de México |
| State/province |
Cuidad de México |
| ZIP/Postal code |
04510 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL10983 |
| Series (1) |
| GSE125801 |
Whole Sequencing Of The Mitochondrial Genome Of Breast Tumor Tissue In Mexican-Mestizo Postmenopausal Women, With Different Body Mass Index |
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