|
| Status |
Public on Apr 09, 2012 |
| Title |
4g-mmg-G |
| Sample type |
RNA |
| |
|
| Source name |
PBL of healthy donor G irradiated with gamma-rays (2.0 Gy) and incubated in modelled microgravity (mmg) for 4h.
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human Peripheral blood lymphocytes (PBL)
|
| Treatment protocol |
Human Peripheral blood lymphocytes (PBL) were isolated by separation on Biocoll density gradient from freshly collected buffy coats from one healthy donor, named G, provided by the Blood Centre of Padova's Hospital. After a overnight incubation, PBL, consisting of peripheral mononuclear cells depleted of monocytes, were irradiated with gamma-rays (2.0 Gy) and then incubated in modelled microgravity (mmg) for 4h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 107 cells by using Trizol® Reagent (Invitrogen) according to the manufacturer’s protocol. Total RNA was quantified using the ND-1000 spectrophotometer (Nanodrop), while RNA integrity and content of microRNAs (%) in each samples were assessed by capillary electrophoresis using the RNA 6000 Nano LabChip and the Small RNA Nano LabChip respectively using the Agilent Bioanalyzer 2100 (Agilent Technologies). Only total RNA samples with R.I.N. (RNA Integrity Number) values of 6, or higher, and the percentage of miRNA < 20% were used for microarray analysis.
|
| Label |
Cy3
|
| Label protocol |
We performed miRNA expression profiling from 200 ng of total RNA and following the miRNA Microarray System protocol v. 1.5 (Agilent Technologies). Total RNA was dephosphorylated and ligated with pCp-Cy3. Unincorporated dyes were removed with MicroBioSpin6 columns (BioRad) . Labeled RNA was hybridized to Agilent miRNA arrays for 22 hours at 55°C using the Agilent's Hybridization Oven that is suited for bubble-mixing and microarray hybridization processes.
|
| |
|
| Hybridization protocol |
Labeled RNA was hybridized to Agilent miRNA arrays for 22 hours at 55°C using the Agilent's Hybridization Oven that is suited for bubble-mixing and microarray hybridization processes. Washing was performed using the Gene Expression Wash Buffer kit (Agilent Technologies) following the manufacturer's instructions.
|
| Scan protocol |
Slides were scanned on an Agilent microarray scanner (model G2565CA) at 100% and 5% sensitivity settings.
|
| Description |
Agilent Feature Extraction software version 10.5.1.1 was used for image analysis.
|
| Data processing |
Only spots with signal minus background flagged as positive and significant (flag=1) were used in the following analysis as “detected” spot while probes with flag equal to 0 were noted as “null”. Probes with less than 60% of detected spots across all arrays were removed from the analysis. Background corrected intensities of replicated spots on each array were averaged. Data were and inter-array normalized with cyclic Lowess (Bolstad BM et al., Bioinformatics (2003), 19(2):185-93) and then log2-transformed.
|
| |
|
| Submission date |
Feb 01, 2010 |
| Last update date |
Apr 09, 2012 |
| 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 |
GPL8227 |
| Series (1) |
| GSE20120 |
Identification of miRNAs involved in cell response to ionising radiation and modeled microgravity |
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