|
| Status |
Public on Mar 01, 2016 |
| Title |
403_11B |
| Sample type |
RNA |
| |
|
| Source name |
Brain tissue
|
| Organism |
Homo sapiens |
| Characteristics |
condition: SIDS
tissue: left central lobe of the brain
|
| Treatment protocol |
Tissue was harvested during autopsy and stored in RNA later in -70C untill analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from 10-20 mg fresh frozen tissues were prepared using the RNeasy mini kits (Qiagen, Valencia, CA) following manufacturer’s instructions. The concentrations of the samples were measured using the NanoDrop Spectrophotometer. The integrity of the samples was assessed using the Agilent 2100 Bioanalyzer. For each sample, 400 ng of total RNA was amplified and labeled. Samples were distributed and processed in a 96-well format and were hybridized to Illumina HumanHT-12 v4 Expression BeadChips. Some samples were up-concentrated using vacuum drying to achieved required concentration/volume.
|
| Label |
biotin
|
| Label protocol |
The Whole-Genome DASL HT Assay begins with conversion of total RNA to cDNA using biotinylated oligo dT and random nonamer primers. The cDNA is then annealed to the DASL Assay Pool probe groups. Probe groups contain oligonucleotides specifically designed to interrogate each target sequence in the transcripts. These probes span about 50 bases, making it possible to profile partially degraded RNA
|
| |
|
| Hybridization protocol |
The assay probe sets consist of an upstream oligo containing a gene-spesific sequence and a universal PCR primer sequence at the 5’ end, and a downstream oligo containing a gene-specific sequence and a universal PCR primer sequence at the 3’. The upstream oligo hybridizes to the targeted cDNA site, and then extends and ligates to its corresponding downstream oligonucleotide to create a PCR template that can be amplified with universal PCR primers.
|
| Scan protocol |
Following washing and staining, the BeadChips were imaged using the Illumina iScan Reader to measure fluorescence intensity at each probe.
|
| Description |
Tissue from left central lobe of the brain
|
| Data processing |
After scanning, the iScan software extracts signal intensities and saves all files for each BeadChip. Raw data is imported to Illumina’s GenomeStudio software V2011.1, Gene Expression module v. 1.9.0. Background correction with bgAdjust.affy in lumi R/Bioconductor package. Quantile normalization, where the three tissue types where quantile normalized separately.
|
| |
|
| Submission date |
Jun 30, 2015 |
| Last update date |
Mar 01, 2016 |
| Contact name |
Linda Ferrante |
| E-mail(s) |
[email protected]
|
| Phone |
0047 99711187
|
| Organization name |
Norwegian institute of public health
|
| Street address |
Gaustadalle 30
|
| City |
Oslo |
| ZIP/Postal code |
0373 |
| Country |
Norway |
| |
|
| Platform ID |
GPL14951 |
| Series (1) |
|
