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| Status |
Public on Jan 11, 2023 |
| Title |
PL.DB_13_1 |
| Sample type |
RNA |
| |
|
| Source name |
neuroretinal homogenate
|
| Organism |
Mus musculus |
| Characteristics |
tissue: neuroretinal homogenate
mouse group: diabetic sample (yellow white)
treatment: eyedrops with vehicle treatment
|
| Treatment protocol |
At the age of 10 weeks, sitagliptin [sitagliptin phosphate monohydrate (Y0001812, Merck KGaA, Darmstadt, Germany) eyedrops (10 mg/mL; n=10), and vehicle [phosphate buffered saline (PBS)] eyedrops (n=10) were randomly administered twice per day directly onto the superior corneal surface of each eye of diabetic mice with the aid of a micropipette (5 µL). On day 15, animals (12 weeks of age) received one drop of sitagliptin or vehicle 1h before euthanasia. 10 non-diabetic mice matched by age were used as control group.
|
| Growth protocol |
Eyes were rapidly enucleated and the neuroretinas were dissected. The neuroretina of each animal was directly stored in an empty tube at -80ºC for transcriptomic experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The neuroretinas were introduced in individual tubes with 140 µL of TRIzol reagent (15596018, InvitrogenTM, Carlsbad, CA, USA) until RNA extraction. For the RNA extraction, neuroretinas were treated with DNase (18068015, ThermoFisher Scientific, Waltham, MA, USA) to remove genomic contamination and were purified on RNeasy MinElute column (74106, Qiagen, Hilden, Germany). After supernatant elimination, RNA sediment was obtained and resuspended in 30 µL of RNAse free water (AM9937, ThermoFisher Scientific, Waltham, MA, USA). An Agilent 2100 Bioanalyzer and a Nanodrop spectrophotometer were used for samples integrity and quantity respectively.
|
| Label |
Biotinylated ds-cDNA
|
| Label protocol |
Biotinylated ds-cDNA were prepared according to the standard Affymetrix (ThermoFisher) protocol from 10 ng total RNA (GeneChip 3' IVT Pico Kit Manual Workflow, user guide).
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| |
|
| Hybridization protocol |
Following fragmentation, 5,5ug of ds-cDNA were hybridized for 16 hr at 45C on GeneChip Clariom S mouse array cartridge. Array cartridge was washed and stained in the Affymetrix GeneChip System 3000
|
| Scan protocol |
Array cartridge was scanned using the scanner GCS3000 (7G with autoloader)
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| Description |
GeneChip 3' IVT Pico Kit. Affymetrix GeneChip System 3000scanned using the scanner GCS3000 (7G with autoloader)
|
| Data processing |
Raw expression values were obtained directly from .CEL files and were processed using the RMA method in oligo R package to get normalized expression values (log2 scale) summarized at the gene level. Different quality checks (boxplots, hierarchical clustering and principal component analyses (PCAs)) were performed before and after normalization to ensure that samples were suitable for differential expression analysis. A complete quality report was obtained using arrayQualityMetrics R package. One sample was identified as a putative outlier during quality control and was therefore excluded from the analysis prior to normalization. The analysis to select differentially expressed genes was based on adjusting a linear model with empirical Bayes moderation of the variance. Because batch effects were detected, a batch factor was included in the linear model used for differential expression analysis. P-values are adjusted to obtain strong control over the false discovery rate using the Benjamini and Hochberg method. Statistical significance was set at 0.05 for adjusted p-values. Statistical language R (version 3.6.1) and Bioconductor associated packages were used to process and analyze the data.
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| |
|
| Submission date |
Nov 30, 2022 |
| Last update date |
Jan 11, 2023 |
| Contact name |
Patricia Bogdanov |
| E-mail(s) |
patricia.bogdanov@vhir.org
|
| Organization name |
Vall d'Hebron Research Institute
|
| Department |
Diabetes Research and Metabolism Unit
|
| Lab |
Collserola Building, Laboratory 144
|
| Street address |
Passeig de la Vall d'Hebron 119-129 Collserola Building. Lab 144
|
| City |
Barcelona |
| State/province |
Barcelona, Spain |
| ZIP/Postal code |
08035 |
| Country |
Spain |
| |
|
| Platform ID |
GPL23038 |
| Series (1) |
| GSE219084 |
Transcriptomic analysis reveals retinal neuromodulation by sitagliptin in an experimental model of diabetic retinopathy |
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