|
| Status |
Public on Jul 10, 2020 |
| Title |
Diabetic.9_versus_pool of non-diabetic |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
non-diabetic skin
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
Sex: male
tissue: skin
disease state: non-diabetic
|
| Treatment protocol |
To obtain type1 diabetic mice, six week-old C57Bl/6 male mice were intraperitoneally injected with 160 mg/kg streptozotocin (Sigma-Aldrich, St. Louis, MO) in 0.05M Na citrate pH 4.5. Seven days after streptozotocin administration, blood glucose levels were measured, and mice with glycaemia above 250 mg/dL were included in the study. Three weeks later, glycaemia was checked again on selected mice, and diabetic mice were sacrificed immediately before collecting their skin.
|
| Growth protocol |
Mice were purchased form Charles River Laboratories (Calco, Lecco, Italy), and maintained on a standard diet. Food and water were given ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Back skin biopsies were collected and snap-frozen. For RNA extraction, frozen biopsies were maintained in liquid nitrogen and crushed to powder with a mortar and pestle. The frozen powder was poured into Trizol (Invitrogen), and RNA extracted following manufacturer’s instructions.
|
| Label |
Hy3
|
| Label protocol |
1.25 ug total RNA was labeled with Hy3, Hy5 resepctively according to the manufacturer's protocol (miRCURY LNA microRNA Array Power labeling kit, EXIQON).
|
| |
|
| Channel 2 |
| Source name |
diabetic skin
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6
Sex: male
tissue: skin
disease state: diabetic
|
| Treatment protocol |
To obtain type1 diabetic mice, six week-old C57Bl/6 male mice were intraperitoneally injected with 160 mg/kg streptozotocin (Sigma-Aldrich, St. Louis, MO) in 0.05M Na citrate pH 4.5. Seven days after streptozotocin administration, blood glucose levels were measured, and mice with glycaemia above 250 mg/dL were included in the study. Three weeks later, glycaemia was checked again on selected mice, and diabetic mice were sacrificed immediately before collecting their skin.
|
| Growth protocol |
Mice were purchased form Charles River Laboratories (Calco, Lecco, Italy), and maintained on a standard diet. Food and water were given ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Back skin biopsies were collected and snap-frozen. For RNA extraction, frozen biopsies were maintained in liquid nitrogen and crushed to powder with a mortar and pestle. The frozen powder was poured into Trizol (Invitrogen), and RNA extracted following manufacturer’s instructions.
|
| Label |
Hy5
|
| Label protocol |
1.25 ug total RNA was labeled with Hy3, Hy5 resepctively according to the manufacturer's protocol (miRCURY LNA microRNA Array Power labeling kit, EXIQON).
|
| |
|
| |
| Hybridization protocol |
2-color hybridization was performed using Exiqon miRCURY LNA microRNA array, 7th generation [miRBase v18]. 1.25ug of each labeled RNA were co-hybridized at 53°C for 16 hours. Hybridization and washing steps were performed on the HS 400 PRO hybridization station (Tecan).
|
| Scan protocol |
MicroRNA arrays were scanned using the GenePix 4100A microarray scanner and the GenePix Pro GenePix Pro 6.1.0.4 software (Molecular Devices).
|
| Description |
microRNA expression levels in the skin of a Type 1 mouse model of diabetes
|
| Data processing |
The obtained signal intensities were analyzed by using the limma package (Bioconductor 2.5 on R 2.10.1 ). Background correction (normexp; cut-off=10) and within-array normalization (global LOESS) was performed. Differential expression and statistical significance were assessed for each miRNA species across all arrays using linear model fit and empirical Bayes method (lmFit, eBayes), taking into account the dye-swap. Spots showing intensities < 3 SNR (for Hy3 and Hy5) OR displaying diameters >130 or <70µm were considered as bad quality spot. The calling of a miRNA failed if 2 or more of the 4 replicates present per miRNA species were bad quality spots and if this occurred on 4 or more arrays.
Processed data file includes normalized log2 ratios representing diabetic/non-diabetic. P value adjusted for False Discovery Rate (Benjamini and Hochberg's method).
|
| |
|
| Submission date |
Sep 17, 2019 |
| Last update date |
Jul 11, 2020 |
| Contact name |
Fabio Martelli |
| E-mail(s) |
fabio.martelli@grupposandonato.it
|
| Phone |
+390252774533
|
| Organization name |
IRCCS-Policlinico San Donato
|
| Lab |
Molecular Cardiology
|
| Street address |
via morandi 30
|
| City |
Milan |
| ZIP/Postal code |
20097 |
| Country |
Italy |
| |
|
| Platform ID |
GPL17107 |
| Series (1) |
| GSE137605 |
Dysregulation of microRNA expression in diabetic skin |
|
