|
| Status |
Public on Feb 21, 2024 |
| Title |
EOCRC13-P3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Colon carcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Early Onset Colorectal Cancer
tissue: colorectal carcinoma
tissue source: Resection
tissue type: formalin fixed paraffin embedded (FFPE) tissue
flow sort content: Human tumor
|
| Growth protocol |
Fresh tumor samples were flash-frozen and maintained at -80 degrees C. Formalin fixed paraffin embedded (FFPE) tissues were stored at room temperature
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Excess paraffin was removed from each FFPE sample with a scalpel from either side of scrolls then processed according to our published methods. We used one to three 50 µm scroll(s) from each FFPE tissue block to obtain sufficient numbers of intact nuclei for sorting and molecular assays. Frozen tissue samples were minced in the presence of NST buffer and DAPI according to published protocols. Nuclei from each sample, FFPE or frozen tissue, were disaggregated then filtered through a 40 μm mesh prior to flow sorting with an Influx or Aria III cytometer (Becton-Dickinson, San Jose, CA) with ultraviolet excitation and DAPI emission collected at >450 nm.
|
| Label |
Cy3
|
| Label protocol |
DNAs from frozen tissue and FFPE samples were treated with DNAse 1 prior to Klenow-based labeling. High molecular weight templates were digested for 30 minutes while DNAs from FFPE samples were digested for only 1 minute. In each case 1 µl of 10x DNase 1 reaction buffer and 2 μl of DNase 1 dilution buffer were added to 7 μl of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. Sample and reference templates were then labeled with Cy-5 dUTP and Cy3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols (22). All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to 400k CGH arrays (Agilent Technologies, Santa Clara, CA)
|
| |
|
| Channel 2 |
| Source name |
Reference pooled 46XX
|
| Organism |
Homo sapiens |
| Characteristics |
reference: Normal female genome
|
| Growth protocol |
Fresh tumor samples were flash-frozen and maintained at -80 degrees C. Formalin fixed paraffin embedded (FFPE) tissues were stored at room temperature
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Excess paraffin was removed from each FFPE sample with a scalpel from either side of scrolls then processed according to our published methods. We used one to three 50 µm scroll(s) from each FFPE tissue block to obtain sufficient numbers of intact nuclei for sorting and molecular assays. Frozen tissue samples were minced in the presence of NST buffer and DAPI according to published protocols. Nuclei from each sample, FFPE or frozen tissue, were disaggregated then filtered through a 40 μm mesh prior to flow sorting with an Influx or Aria III cytometer (Becton-Dickinson, San Jose, CA) with ultraviolet excitation and DAPI emission collected at >450 nm.
|
| Label |
Cy-5
|
| Label protocol |
DNAs from frozen tissue and FFPE samples were treated with DNAse 1 prior to Klenow-based labeling. High molecular weight templates were digested for 30 minutes while DNAs from FFPE samples were digested for only 1 minute. In each case 1 µl of 10x DNase 1 reaction buffer and 2 μl of DNase 1 dilution buffer were added to 7 μl of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. Sample and reference templates were then labeled with Cy-5 dUTP and Cy3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols (22). All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to 400k CGH arrays (Agilent Technologies, Santa Clara, CA)
|
| |
|
| |
| Hybridization protocol |
Labeled DNA was hybridized without ozone scavenger and chips washed according to manufacture's protocol for 40 hours in a rotating 65°C oven.
|
| Scan protocol |
All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 11.0 using default settings.
|
| Data processing |
Data was extracted from the TIFF files using Agilent FE 11. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm (ADM2)[23]. The latter identifies all aberrant intervals in a given sample with consistently high or low log ratios based on the statistical score derived from the average normalized log ratios of all probes in the genomic interval multiplied by the square root of the number of these probes. This score represents the deviation of the average of the normalized log ratios from its expected value of zero and is proportional to the height h (absolute average log ratio) of the genomic interval, and to the square root of the number of probes in the interval.
|
| |
|
| Submission date |
Aug 08, 2023 |
| Last update date |
Feb 21, 2024 |
| Contact name |
Michael Thomas Barrett |
| E-mail(s) |
barrett.michael@mayo.edu
|
| Phone |
480-301-6736
|
| Organization name |
Mayo Clinic Arizona
|
| Department |
Molecular Pharmacology and Experimental Therapeutics
|
| Street address |
13400 East Shea Boulevard
|
| City |
Scottsdale |
| State/province |
AZ |
| ZIP/Postal code |
85259 |
| Country |
USA |
| |
|
| Platform ID |
GPL19387 |
| Series (1) |
| GSE240339 |
Genomic Landscape of Diploid and Aneuploid Microsatellite Stable Early Onset Colorectal Cancer |
|
