|
| Status |
Public on Apr 01, 2020 |
| Title |
PFP-Healthy-13 |
| Sample type |
RNA |
| |
|
| Source name |
Platelets-free Plasma Healthy control subject 13
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Healthy control subject
tissue: Platelets-free Plasma
|
| Treatment protocol |
Platelets-free plasma was obrtained from peripheral blood samples in citrate tubes via sequantial centrifugation. Plasma samples were divided in to aliquots and stored at-80°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from freshly isolated platelets-free plasma using TRIzol LS (Invitrogen S.R.L, Italy) reagent following manufacturer instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) RNA samples were stored at -80°C until use.
|
| Label |
Cy3
|
| Label protocol |
100 ng RNA was dephosphorylated and labeled by miRNA Complete Labeling and Hyb Kit (Agilent) according to the manufacturer's instructions.
|
| |
|
| Hybridization protocol |
The dried and labeled miRNA sample was resuspended in 18 ul nuclease-free water, 4.5 ul 10X GE Blocking Agent (Agilent) and 22.5 ul 2X Hi-RPM hybridization buffer (Agilent), followed by heating at 100°C for 5 minutes. Upon cooling in ice water bath for 5 mintues, the mixture was hybridized to Human miRNA Microarray based on Sanger miRbase (release 9.1). (G4471A) for 20 hours at 55°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minutes at room temperature with GE Wash Buffer 1 (Agilent) and 5 minutes with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x15k Agilent- 016436 Human miRNA Microarray 1.0 G447A array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%)
|
| Description |
Circulating Extracellular miRNAS expression profiling
C_PFP_38
|
| Data processing |
The scanned TIFF images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (Protocol: miRNA-v1_95_May07 Grid: 016436_D_20070426 ) to obtain background subtracted and spatially detrended Processed Signal intensities. Background-subtracted values were normalized between arrays by Quantile algorithm, included in the R package AgiMicroRna (López-Romero, P. BMC Genomics. 2011.).
|
| |
|
| Submission date |
Feb 12, 2020 |
| Last update date |
Apr 03, 2020 |
| Contact name |
Giacomo Bagni |
| E-mail(s) |
[email protected]
|
| Organization name |
Università degli studi di Firenze
|
| Department |
Department of Experimental and Clinical Medicine
|
| Lab |
Arcangeli
|
| Street address |
Viale GB Morgagni 50
|
| City |
Florence |
| ZIP/Postal code |
50134 |
| Country |
Italy |
| |
|
| Platform ID |
GPL9081 |
| Series (1) |
| GSE145191 |
Circulating miRNome expression profiling in Behçet's disease |
|
