|
| Status |
Public on Sep 30, 2011 |
| Title |
C4_control |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control C4
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: unaffected with parkinson´s disease
sex: Male
age: 71 yrs
tissue: PBMCs
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 16 ml of whole blood was collected by venipuncture into two BD Vacutainer CPT glass tubes (Becton-Dickinson, Franklin Lakes, USA). Whithin two hours of blood collection, peripheral blood mononuclear cells (PBMCs) were isolated from the CPT tubes by centrifugation (30 min, 2800 rpm, room temperature) and washed twice with PBS (15 min, 1300 rpm, 4ºC). Total RNA containing small RNAs was extracted from PBMCs using the miRNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, aliquoted and stored at -80ºC. All samples passed extensive quality controls (e.g. OD260/OD280 between 1.8 and 2.1 in the Nanodrop ND1000 and RIN (RNA Integrity Number) > 7 in the Agilent 2100 Bioanalyser).
|
| Label |
Hy3
|
| Label protocol |
RNA sample was sent to Exiqon (Vedbaek, Denmark) for miRNA profiling. 1µg of total RNA was labeled with the miRCURY Hy3 power labeling kit.
|
| |
|
| Channel 2 |
| Source name |
Common reference pool
|
| Organism |
Homo sapiens |
| Characteristics |
sample: Pool composed of 39 samples (the 32 samples under study and 7 additional samples which were later excluded from differential expression analysis due to array quality control problems).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Same protocol as channel 1
|
| Label |
Hy5
|
| Label protocol |
RNA samples were sent to Exiqon (Vedbaek, Denmark) for miRNA profiling. 1µg of total RNA from each of the 39 samples was pooled and labeled with the miRCURY Hy5 power labeling kit.
|
| |
|
| |
| Hybridization protocol |
RNA samples were sent to Exiqon (Vedbaek, Denmark) for miRNA profiling. Briefly, the Hy3-labeled sample and the Hy5-labeled common reference pool were co-hybridized to the miRCURYTM locked nucleic acid (LNA) array (version 10.0) using a Tecan (Austria) HS4800 hybridization station.
|
| Scan protocol |
Arrays were scanned with an Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and stored in an ozone-free environment in order to prevent potential bleaching of the fluorescent dyes.
|
| Description |
Image analysis was carried out using the ImaGene 7.0 software (BioDiscovery, Inc., USA) and the resulting ImaGene output files were analyzed using the Limma package (Smyth GK Stat Appl Genet Mol Biol. 2004; 3:Article3) implemented using R freeware (http://cran.r-project.org/).
|
| Data processing |
Background correction was performed using the normexp method (with offset=10). Intensities of good quality spots were then subjected to within-array (loess method) normalization. Intensities lower than 1.5 times the median signal intensity of the respective slide and label were discarded. The calling of a miRNA on a particular array fails if two or more of the four replicated capture probes for this miRNA are flagged as bad quality spots by the image analysis software. Differential expression between controls and PD patients was assessed using linear regression analysis and empirical Bayes methods implemented in the Limma package.
|
| |
|
| Submission date |
Jun 15, 2009 |
| Last update date |
Sep 30, 2011 |
| Contact name |
Sofia A Oliveira |
| E-mail(s) |
[email protected]
|
| Phone |
+351 217 999 411
|
| Fax |
+351 217 999 412
|
| Organization name |
Instituto de Medicina Molecular
|
| Department |
Edificio Egas Moniz
|
| Lab |
P1C-76
|
| Street address |
Av. Prof Egas Moniz
|
| City |
Lisbon |
| State/province |
Portugal |
| ZIP/Postal code |
1649-028 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL7722 |
| Series (1) |
| GSE16658 |
microRNA (miRNA) expression profiling of PBMCs of Parkinson's disease (PD) patients and controls |
|
