|
| Status |
Public on Dec 31, 2017 |
| Title |
FAMI patient N. 21 (B) |
| Sample type |
RNA |
| |
|
| Source name |
Platelets from patient with acute MI within 6 hours of the onset of symptoms
|
| Organism |
Homo sapiens |
| Characteristics |
age (years): 74
gender: male
bmi: 29.75
n. diseased vessels: 2
ck peak: 488.00
tnl on admission (pg/ml): 1.65
crp on admission (mg/dl): 3.67
il-6 on admission (ng/dl): 2.08.
|
| Biomaterial provider |
Cardio-Thoracic Unit of San Raffaele Hospital (Milan, Italy).
|
| Treatment protocol |
Patients were all with the same clinical presentation: acute MI within 6 hours of the onset of symptoms, without any previous evidence of coronary disease, except for the retrospective report of possible episodes of angina in the previous week and potentially eligible for thrombolysis or other coronary reperfusion strategie.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Peripheral blood (40 ml) from patients was collected in EDTA, and platelets were isolated within 1 hour by differential centrifugation. Purity of platelet preparations was checked with a Cell-Dyn Coulter counter (Abbot Diagnostics, Abbott Park). A fraction of platelets was resuspended in TRIzol reagent (Invitrogen) for total RNA isolation according to the manufacturer's guidelines. Total RNA was quantified using the ND-1000 spectrophotometer (Nanodrop, Wilmington, DE), while RNA integrity was assessed by capillary electrophoresis using the RNA 6000 Nano LabChip (Agilent Technologies, Palo Alto, CA). Platelet purification was also checked after total RNA extraction by quantitative RT-PCR on the platelet specific marker, integrin alpha 2b, and the leukocyte specific marker T cell receptor (TCR). Good samples, used for further analysis and experiments, showed < 1% TCR purity level.
|
| Label |
Cy5
|
| Label protocol |
We performed microarray experiments using the CodeLink Expression Bioarray System (GE Healthcare). Total RNA (1.5 µg) was amplified and biotin-labeled using the CodeLink Expression Assay Reagent Kit following the manufacturer’s protocol. Before amplification, mRNA control spikes were added to all tubes. These controls were used to verify the quality of the process. Biotin-labeled aRNA (10 µg) was fragmented and loaded into a CodeLink Human Whole Genome Bioarray, containing about 55,000 human oligonucleotide gene probes, matching more than 45,000 human genes and transcript targets
|
| |
|
| Hybridization protocol |
Arrays were hybridized for 21 hours at 37°C at 300 rpm on a Thermomixer comfort (Eppendorf) and washed according to manufacturer’s protocol. The bioarrays were stained with incubation in 3.4 mL of streptavidin-Cy5 solution for 30 minutes, washed, and dried by centrifugation at 640 rpm.
|
| Scan protocol |
Images were obtained with ScanArray Lite (PerkinElmer) scanner, using the ScanArray Express software (PerkinElmer), with a 635 nm laser, maintaining constant laser power (85%) and constant photomultiplier gain (55%).
|
| Description |
Images were analyzed using CodeLink expression software version 4.2, and raw data analysis was exported to GeneSpring format (tab-delimied text format).
|
| Data processing |
Raw intensity signals were analyzed with the GeneSpring GX 7.3.1 software (Agilent Technologies). Normalization steps included: data transformation, setting measurements less than 0.01 to 0.01; per chip and per gene median polishing; per gene normalization to specific samples (i.e. normal controls). Before performing the statistical analysis we filtered genes with the “present” or “marginal” quality flag values (automatically assigned to each spot by the CodeLink software, according to a signal to noise computation) in 28 out of 38 conditions for platelet data.
|
| |
|
| Submission date |
Oct 05, 2010 |
| Last update date |
Dec 31, 2017 |
| 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 |
GPL2895 |
| Series (2) |
| GSE24519 |
Gene expression profiling of patients affected by first acute myocardial infarction (FAMI) |
| GSE24591 |
Gene and microRNA expression profiling of patients affected by first acute myocardial infarction (FAMI) |
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