|
| Status |
Public on Oct 01, 2012 |
| Title |
DNA normozospermic 103 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
genomic DNA extracted from lymphocytes of a normozospermic subject
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Male
phenotype: normozoospermia
cell type: lymphocyte
cnv count: 1
cnv code: 36.A
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA extracted using QIAamp DNA mini kit or Salting out procedure following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
300ng of test DNA and control DNA were double-digested with RsaI and AluI (Promega) for 1 hour at 37°C. After digestion, samples were incubated at 65°C for 20 minutes to inactivate the enzymes, then labeled by random priming (Agilent Technologies) for 2 hours using Cy5-dUTP for the test DNA and Cy3-dUTP (Agilent Technologies) for the control DNA. Labeled DNAs were incubated at 65°C for 10 minutes and then purified with Microcon YM-30 filter units (Millipore, Billerica, USA). Every purified sample was brought to a total volume of 9.5µl in 1XTE (pH 8.0, Promega), and yield and specific activity were determined for each sample using a NanoDrop ND-1000 UV-VIS Spectrophotometer (Labtech International LTD). The appropriate cyanine 5- and cyanine 3-labeled samples were combined in a total volume of 16μl.
|
| |
|
| Channel 2 |
| Source name |
genomic DNA extracted from lymphocytes of the reference normozoospermic control subject
|
| Organism |
Homo sapiens |
| Characteristics |
phenotype: normozoospermic
gender: male
cell type: lymphocyte
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA extracted using QIAamp DNA mini kit or Salting out procedure following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
300ng of test DNA and control DNA were double-digested with RsaI and AluI (Promega) for 1 hour at 37°C. After digestion, samples were incubated at 65°C for 20 minutes to inactivate the enzymes, then labeled by random priming (Agilent Technologies) for 2 hours using Cy5-dUTP for the test DNA and Cy3-dUTP (Agilent Technologies) for the control DNA. Labeled DNAs were incubated at 65°C for 10 minutes and then purified with Microcon YM-30 filter units (Millipore, Billerica, USA). Every purified sample was brought to a total volume of 9.5µl in 1XTE (pH 8.0, Promega), and yield and specific activity were determined for each sample using a NanoDrop ND-1000 UV-VIS Spectrophotometer (Labtech International LTD). The appropriate cyanine 5- and cyanine 3-labeled samples were combined in a total volume of 16μl.
|
| |
|
| |
| Hybridization protocol |
After sample denaturation and pre-annealing with 5µl of Human Cot-1 DNA (Invitrogen, Carlsbad, CA), hybridization was performed at 65°C with shaking for 24 hours. After two washing steps, the array was analyzed through the Agilent scanner and the Feature Extraction software (v10 1.1.1).
|
| Scan protocol |
Scanned on an Agilent G2539A scanner.
Images were quantified using Agilent Feature Extraction Software (version 10 1.1.1).
|
| Description |
normozoospermia
|
| Data processing |
Agilent Feature Extraction Software (version 10 1.1.1) was used for background subtraction and LOWESS normalization. Graphical overview was obtained using the DNA Analytics (v4.0.73). All the array experiments were analysed using the ADM-2 algorithm at threshold 5. Text aberration summaries were analyzed to calculate the average log2 ratios of each Copy Number Variation and we considered at least 3 adjacent oligonucleotides needed for a reliable call. The positions of oligomeres refer to the Human Genome March 2006 assembly (hg18).
|
| |
|
| Submission date |
May 11, 2012 |
| Last update date |
Oct 01, 2012 |
| Contact name |
Chiara Chianese |
| E-mail(s) |
chiara.chianese@live.it
|
| Organization name |
Univerity of Florence
|
| Street address |
viale Pieraccini. 6
|
| City |
Florence |
| ZIP/Postal code |
50139 |
| Country |
Italy |
| |
|
| Platform ID |
GPL15560 |
| Series (1) |
| GSE37948 |
X-linked CNV burden and male infertility |
|
