|
| Status |
Public on Sep 01, 2016 |
| Title |
ALI |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ALI_enriched methylated DNA
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: RMS tumor tissue
tumor stage: stage III
|
| Growth protocol |
fresh or frozen tumor tissue
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from RMS tumor biopsies using Trizol Reagent (Life Technologies) after RNA extraction, following the manufacturer’s protocol. The commercially available Qiamp DNA mini Kit (Qiagen) was used to DNA purification. Four ug of genomic DNA were fragmented using a sonicator, purified using Mini-Elute coloumns (Qiagen) and the amount of double-strand DNA (dsDNA) was measured using Qubit instrument. The success of fragmentation was evaluated using the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). The MethylMiner Methylated DNA enrichment kit (Invitrogen, Life technologies) was used to the enrichment and fractioned of methylated dsDNA starting from 2ug of fragmented whole genomic DNA. Ten ng of methylated dsDNA for each sample was amplified using Whole genome Amplification (WGA, Sigma-Aldrich).
|
| Label |
cy5
|
| Label protocol |
The control genomic DNA and enriched methylated dsDNA were labeled with Cy3 and Cy5 dye respectively using Agilent Genomic DNA labeling kit PLUS (Agilent)according to the manufacturer’s instructions
|
| |
|
| Channel 2 |
| Source name |
ALI_genomic DNA
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: RMS tumor tissue
tumor stage: stage III
|
| Growth protocol |
fresh or frozen tumor tissue
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from RMS tumor biopsies using Trizol Reagent (Life Technologies) after RNA extraction, following the manufacturer’s protocol. The commercially available Qiamp DNA mini Kit (Qiagen) was used to DNA purification. Four ug of genomic DNA were fragmented using a sonicator, purified using Mini-Elute coloumns (Qiagen) and the amount of double-strand DNA (dsDNA) was measured using Qubit instrument. The success of fragmentation was evaluated using the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). The MethylMiner Methylated DNA enrichment kit (Invitrogen, Life technologies) was used to the enrichment and fractioned of methylated dsDNA starting from 2ug of fragmented whole genomic DNA. Ten ng of methylated dsDNA for each sample was amplified using Whole genome Amplification (WGA, Sigma-Aldrich).
|
| Label |
cy3
|
| Label protocol |
The control genomic DNA and enriched methylated dsDNA were labeled with Cy3 and Cy5 dye respectively using Agilent Genomic DNA labeling kit PLUS (Agilent)according to the manufacturer’s instructions
|
| |
|
| |
| Hybridization protocol |
The control genomic DNA and methylated dsDNA labeled with Cy3 and Cy5 dye were competitively hybridized to Human DNA Methylation microarrays platforms.The hybridization was carried out at 67°C for 40 hours in a hybridization oven rotator (Agilent). The arrays were washed with Agilent ChiP-on-chip wash buffers as suggest by the supplier.
|
| Scan protocol |
Slides were scanned on an Agilent microarray scanner (model G2565CA), and Agilent Feature Extraction software version 10.7.3.1 was used for image analysis.
|
| Description |
Enriched methylated DNA (cy5) and genomic DNA (cy3) of each sample were competitively hybridizated to Human DNA Methylation microarrays platforms (Agilent)
|
| Data processing |
Probe log ratio signals were Loess and linear normalized .Inter-array normalization was performed with quantile normalization. Feature Extraction Software (Agilent) provides spot quality measures in order to evaluate the quality and the reliability of the hybridization data. In particular, flag glsFound and “rlsFound”(set to 1 if the spot had an intensity value that was significantly different from the local background or to 0 in any other case) was used to filter out unreliable probes: flag equal to 0 was to be noted as not available (NA). In order to make more robust and unbiased statistical analyses, probes with a high proportion of NA values were removed from the dataset. Twenty-five percent of NA was used as the threshold in the filtering process, obtaining a total of 90,591 available probes.
|
| |
|
| Submission date |
Mar 24, 2015 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Gerolamo Lanfranchi |
| E-mail(s) |
stefano.cagnin@unipd.it
|
| Phone |
+39-0498276219
|
| Organization name |
University of Padova
|
| Department |
CRIBI - Biotechnology Center and Biology Department
|
| Lab |
Functional Genomics Lab
|
| Street address |
Via U. Bassi, 58/B
|
| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19930 |
| Series (1) |
| GSE67201 |
DNA methylation profiling of rhabdomyosarcoma tumor samples |
|
