|
| Status |
Public on Dec 21, 2011 |
| Title |
Pt032B Distal mucosa_Tongue |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Distal mucosa
|
| Organism |
Homo sapiens |
| Characteristics |
main site: Tongue
histology: Non dysplastic
smoke habit: Smoker
age (years): 64
dna index: 1
gender: Male
dna amplification kit: Sigma
|
| Treatment protocol |
Samples were processed to obtain DAPI stained nuclei suspensions following the method of Otto et al., (Methods Cell Biol., 1994 Academic Press San Diego; 211-217) with modifications. High resolution DNA flow cytometry of these samples was used to obtain DNA content histograms and to evaluate the DNA Index (DI), as previously described in details (Donadini et al., 2010, Cell. Oncol. 32, 373-383). DNA diploid controls were represented by gender-specific human lymphocytes. The mean CV of the corresponding DNA diploid G0-G1 peaks was 1.2±0.2%. The DNA diploid (DI=1) and aneuploid sublines were sorted using a Cyflow Space FCM equipped with a PPCS unit (Partec GmbH) at the purity of about 99%.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted using ArchivePure DNA (5-Prime) with some modifications: after protein precipitation the DNA was purified by phenol-chloroform extraction and collected by ethanol precipitation using 20 µg of glycogen (20 mg/ml). DNA quality was assessed by agarose gel and ND-1000 spectrophotometer (NanoDrop Technologies).
|
| Label |
Cy5
|
| Label protocol |
DNA was amplified using GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich) (DNA= 50ng) or Ovation WGA System kit (Nugen Technologies) (DNA<50ng). We followed the "Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol”, v. 5.0 (Agilent Technologies). For each array, amplified test and reference DNAs (2 µg each) were labeled by Bioprime Labeling Kit (Invitrogen, Paisley, UK). Briefly, amplified DNA was labeled with Cy3-dUTP or Cy5-dUTP (Perkin Elmer) (reference and test samples, respectively) using the Random-Primed Bioprime DNA Labeling kit (Invitrogen Life Technologies). Unincorporated nucleotides were removed on Microcon YM-30 filters (Millipore) according to the manufacturer’s protocol. Dye incorporations of post-labeling products were measured by ND-1000 spectrophotometer (NanoDrop Technologies) and the parameters that predicted successful hybridization were a minimum Cy3 incorporation of 0.5 pmol/ul and Cy5 incorporation of 0.3 pmol/ul.
|
| |
|
| Channel 2 |
| Source name |
Human Genomic DNA: male Cat G147A Promega, Madison, WI
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Male
|
| Treatment protocol |
Samples were processed to obtain DAPI stained nuclei suspensions following the method of Otto et al., (Methods Cell Biol., 1994 Academic Press San Diego; 211-217) with modifications. High resolution DNA flow cytometry of these samples was used to obtain DNA content histograms and to evaluate the DNA Index (DI), as previously described in details (Donadini et al., 2010, Cell. Oncol. 32, 373-383). DNA diploid controls were represented by gender-specific human lymphocytes. The mean CV of the corresponding DNA diploid G0-G1 peaks was 1.2±0.2%. The DNA diploid (DI=1) and aneuploid sublines were sorted using a Cyflow Space FCM equipped with a PPCS unit (Partec GmbH) at the purity of about 99%.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted using ArchivePure DNA (5-Prime) with some modifications: after protein precipitation the DNA was purified by phenol-chloroform extraction and collected by ethanol precipitation using 20 µg of glycogen (20 mg/ml). DNA quality was assessed by agarose gel and ND-1000 spectrophotometer (NanoDrop Technologies).
|
| Label |
Cy3
|
| Label protocol |
DNA was amplified using GenomePlex Whole Genome Amplification Kit (Sigma-Aldrich) (DNA= 50ng) or Ovation WGA System kit (Nugen Technologies) (DNA<50ng). We followed the "Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol”, v. 5.0 (Agilent Technologies). For each array, amplified test and reference DNAs (2 µg each) were labeled by Bioprime Labeling Kit (Invitrogen, Paisley, UK). Briefly, amplified DNA was labeled with Cy3-dUTP or Cy5-dUTP (Perkin Elmer) (reference and test samples, respectively) using the Random-Primed Bioprime DNA Labeling kit (Invitrogen Life Technologies). Unincorporated nucleotides were removed on Microcon YM-30 filters (Millipore) according to the manufacturer’s protocol. Dye incorporations of post-labeling products were measured by ND-1000 spectrophotometer (NanoDrop Technologies) and the parameters that predicted successful hybridization were a minimum Cy3 incorporation of 0.5 pmol/ul and Cy5 incorporation of 0.3 pmol/ul.
|
| |
|
| |
| Hybridization protocol |
Labeled DNAs were hybridized to Human Genome CGH 105K array platform (Agilent Technologies)following the "Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol”, v. 5.0. Briefly, fluorescent-labeled reference and tumor DNAs (4 µg each) were mixed with 25 µg of human Cot-1 DNA (Invitrogen Life Technologies) and control targets (Agilent). Slides were hybridized in SureHyb gasket (Agilent Technologies) placed in rotisserie (20 RPM rotation speed) in hybridization oven at 65°C for 40 hrs. After hybridization, the slides were washed with Oligo aCGH Wash Buffer 1 (Agilent Technologies) at room temperature for 5 min, with Oligo aCGH Wash Buffer 2 (Agilent Technologies) at 37°C for 1 min, with the Stabilization and Drying Solution (Agilent) for 1 minute at room temperature.
|
| Scan protocol |
Slides were scanned immediately after washing by the Agilent G2565BA Scanner using two color scan setting for 2x105k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel 1 is set to Red & Green, Red PMT is set to 100%, Green PMT is set to 100%) . Microarray performance was assessed by QC metric tool.
|
| Description |
DNA amplification kit: Sigma
aCGH analysis
|
| Data processing |
The scanned TIFF images were processed by Feature Extraction version 9.5.3.1 (Agilent Technologies). Data files were analyzed byAgilent CGH Analytics Software (version 3.5.14) according to ADM-2 algorithm. The quality of each experiment was assessed by QC metric tool.
|
| |
|
| Submission date |
Apr 28, 2011 |
| Last update date |
Dec 21, 2011 |
| Contact name |
PAOLA SCARUFFI |
| E-mail(s) |
[email protected]
|
| Organization name |
"San Martino" Hospital
|
| Lab |
Center of Physiopathology of Human Reproduction
|
| Street address |
LARGO R. BENZI, 10
|
| City |
Genova |
| State/province |
GE |
| ZIP/Postal code |
16132 |
| Country |
Italy |
| |
|
| Platform ID |
GPL4093 |
| Series (1) |
| GSE28906 |
Genomic aberrations in distant fields from oral potentially malignant lesions by high resolution DNA flow cytometry and array-CGH |
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