|
| Status |
Public on Dec 03, 2019 |
| Title |
Skin fibroblasts, Patient4-replica3 |
| Sample type |
RNA |
| |
|
| Source name |
skin fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
condition: Aicardi Goutières Syndrome (AGS)
cell line: P4
race: Indian
tissue: skin
cell type: fibroblast
gender: Female
cell cycle stage: 20% S phase
|
| Treatment protocol |
Untreated samples
|
| Growth protocol |
All fibroblasts were grown in DMEM with 4.5 g/liter glucose, 10% (v/v) fetal calf serum, nonessential amino acids, and antibiotics. Cell cycle distribution in fibroblast cultures was determined by flow cytometry after propidium iodide staining of EtOH 70% fixed resuspended cells, with a BD Biosciences FACSCanto II Flow cytometer. To identify 20% S-phase enriched cell culture, 0.2 million cells/ 10 cm dish were seeded and collected for FACS analysis starting after 30 hours and every 2-3 hours until two days post seeding.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol Reagent (Thermo Fisher Scientific) was used to extract RNA from cell cultures according to the manufacturer’s protocol. Prior to RNA extraction, cell cultures were washed in PBS to remove excess medium then Trizol was added directly on the dish and cells were detached using a cell scraper. RNA quality was tested on UV spectrophotometer and 2100 Agilent Bioanalyzer following the protocol provided by the manufacturer.
|
| Label |
Cy3
|
| Label protocol |
Fluorescent cRNA to hybridize onto microarray was produced by Low Input Quick Amp Labeling Kit (Agilent) according to manufacturer instructions.
|
| |
|
| Hybridization protocol |
After cRNA labeling it was dispersed onto the microarray to perform the hybridization at 65°C for 17 h with 10 rpm rotation. Finally, slides were washed using Wash Buffer Kit (Agilent Technologies) and dried at room temperature.
|
| Scan protocol |
Microarray slides were scanned using G2505C scanner (Agilent Technologies) at 3 µm resolution. Probes features were extracted using the Feature Extraction Software v. 10.7.3.1 with GE_1_Sep09 protocol (Agilent Technologies).
|
| Description |
P4-3
|
| Data processing |
Intra-array normalization was directly performed by the Feature Extraction Software. For each sample, we set probe expression to NA (not available) when flag “Positive and Significant” from Feature Extraction Software was ‘FALSE’. To avoid problems in clustering analysis NA was transformes as 0.1. To normalize data, we used quantile inter-arrays normalization (normalizeQuantiles, limma R package). Data for long non-coding RNAs were excluded for the normalization of coding RNAs. The expression of probes with the same ProbeName was averaged. Microarray data were analysed using the MultiExperiment Viewer (MeV, Ver. 4.8). We used a t-test (from MeV) to identify differentially expressed RNAs between cells derived from AGS patients and WT. P-values were computed using a gene permutation approach and corrected using Bonferroni. We consider a gene differentially expressed when corrected p-value was ≤ 0.05. Differentially expressed genes resulted from each comparison were grouped to identify interesting profiles considering all samples. Gene grouping was performed using the Self Organizing Tree Algorithm (SOTA) implemented in the MultiExperiment Viewer software. Clustering was performed using the Pearson correlation. Genes in specific clusters were classified according to their function using the Reactome database implemented in the WEB-based GEne SeT AnaLysis Toolkit. To calculate FDR corresponding to each pathway it was used the Benjamini and Hochberg correction for multiple tests.
|
| |
|
| Submission date |
Aug 09, 2019 |
| Last update date |
Dec 03, 2019 |
| 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 |
GPL17077 |
| Series (1) |
| GSE135652 |
Gene expression of fibroblasts carrying SAMHD1 mutations or not |
|
