|
| Status |
Public on Aug 26, 2013 |
| Title |
Carotid_media_pDC_1 |
| Sample type |
RNA |
| |
|
| Source name |
Carotid_between media and plaque_pDC-enriched
|
| Organism |
Homo sapiens |
| Characteristics |
age (yrs): 83
gener: male
tissue: Human atherosclerotic plaque
|
| Treatment protocol |
4 mm thick OCT frozen sections were cut at -20°C in a cryostat (Leica CM 3050S) and placed on MembraneSlides (NF 1.0 PEN; Carl Zeiss Micro Imaging, Munich, Germany). Sections were fixed and dehydrated for 10 minutes in ice-cold sterile acetone before LCM (Palm Microlaser Technologies, Zeiss Microscope Palm Robo 4.3, Munich, Germany) to improve tissue catapulting. pDC-enriched and pDC-deficient plaque fractions from the very same plaque slices were isolated by LCM from 6-10 frozen tissue sections, respectively pooled and collected in nuclease and nucleid acid free tubes with an adhesive cap covered with tissue lysis buffer (RLT buffer) containing β-mercaptoethanol to improve RNA yield and minimize RNA degradation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy Micro kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol for extraction of RNA from microdissected cryosections. DNA was removed by using 50U DNase I (Qiagen). RNA samples were speed vacuum centrifuged to increase RNA concentration. Total RNA (500-750pg) of pDC-enriched and pDC-deficient plaque fractions was cDNA transcribed (double strand cDNA synthesis), purified (RNAClean beads) and amplified (Ribo-SPIA amplification process) using the Ovation Pico WTA System V2 (NuGEN Technologies, Bemmel, The Netherlands).
|
| Label |
biotin
|
| Label protocol |
cDNA samples were enzymatically fragmented (input: 3 μg per sample) using the Ovation Pico WTA System V2 and biotin-labeled to the 3-hydroxyl end of the fragmented cDNA using the Encore Biotin Module (NuGEN Technologies).
|
| |
|
| Hybridization protocol |
2.5 μg of labeled cDNA of each sample was applied to Affymetrix GeneChip Human Gene 1.0 ST arrays (Affymetrix, Santa Clara, CA, USA).
|
| Scan protocol |
GeneChips were scanned using the Affymetrix Gene Chip Scanner 7G and feature extraction was done by Gene Chip® Command Console 3.2 (AGCC) software.
|
| Description |
Array4
Gene expression data from inflammatory region within human atherosclerotic plaques obtained from endarterectomy of the carotid
|
| Data processing |
The data were analyzed with R (v.2.13)/Bioconductor (v.2.8) using the package xps (v.1.12.1) and the most up to date .CDF (HuGene-1_0-st-v1.r3.cdf) available from the Affymetrix website at the time. .
The 'HuGene10stv1_Hs_ENTREZG_14.1.0' was used to summarize the probesets to Entrez Genes.
|
| |
|
| Submission date |
Aug 08, 2013 |
| Last update date |
Aug 27, 2013 |
| Contact name |
Marco Manca |
| E-mail(s) |
marco.manca@cern.ch, marco@scimpulse.org
|
| Organization name |
University of Maastricht
|
| Department |
CARIM
|
| Lab |
Experimental Vascular Pathology
|
| Street address |
P. Debyelaan 25
|
| City |
Maastricht |
| State/province |
Limburg |
| ZIP/Postal code |
6229 HX |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL15034 |
| Series (1) |
| GSE49670 |
Expression data from laser capture microdissected human atherosclerotic plaque, rich or void of plasmacytoid dendritic cells |
|
