|
| Status |
Public on Jun 01, 2022 |
| Title |
PSD LCM EndCells_10 |
| Sample type |
RNA |
| |
|
| Source name |
Laser capture miscrodissected endothelial cells from human PSD brain tissue
|
| Organism |
Homo sapiens |
| Characteristics |
gender: F
condition: PSD
|
| Treatment protocol |
Toluidine blue histologically stained pyramidal neurons, glial fibrillary acidic protein-positive (GFAP+)- astrocytes and collagen IV-positive (Coll-IV+)- endothelial cells were isolated from each human case (PSD, PSND and Control) using a PixCell II laser capture microdissection (LCM) system. Toluidine blue histologically stained pyramidal neurons were isolated from each mouse case (BCAS and SHAM) using a PixCell II laser capture microdissection (LCM) system.
|
| Growth protocol |
All samples were snap frozen and stored at -80°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each isolated group of cells using the PicoPure RNA isolation kit
|
| Label |
Biotin
|
| Label protocol |
Target labelled mRNA was prepared following the GeneChip WT Pico amplification protocol (Affymetrix). RNA amplification was achieved using low-cycle PCR followed by amplification using T7 in vitro transcription. The cRNA was converted to biotinylated sense-strand DNA hybridisation targets.
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| |
|
| Hybridization protocol |
Approximately 5.5 μg amplified cDNA was fragmented, labelled and hybridised to Affymetrix Clariom S Array chips for 16 h at 45 °C in a rotating oven at 60 rpm
|
| Scan protocol |
Using the Fluidics Station 400, and Gene Chip Operating System, the hybridisation cocktail was removed, and a series of stringency washing steps followed to remove any unbound DNA, before each microarray was stained. Each microarray chip was scanned using the GeneChip 3000 scanner to determine the fluorescence intensity of hybridised transcripts.
|
| Description |
Gene expression data from PSD endothelial cells
|
| Data processing |
Transcription Analysis Console (TAC) was used for quality control and analysis of the data, the .CEL files were imported into TAC version 4.0.1 (Applied Biosystems, USA) and normalised to the median of all genes, prior to statistical analysis, to identify differentially expressed genes (DEGs). Transcripts were defined as being significantly differentially expressed if they showed a p-value of < 0.05 (unpaired t-test). DEGs were classified according to their biological function using the Database for Annotation Visualisation and Integrated Discovery (DAVID) using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify specific pathways while Functional Annotation Clustering (FAC) was used to cluster together functionally similar genes. In the case of a significant result, the p value was adjusted for multiple testing using the Bonferroni correction method and considered significant if p < 0.05
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| |
|
| Submission date |
Oct 29, 2021 |
| Last update date |
Jun 01, 2022 |
| Contact name |
Rachel Waller |
| E-mail(s) |
[email protected]
|
| Organization name |
The University of Sheffield
|
| Department |
Neuroscience
|
| Street address |
SITraN, 385A Glossop Road
|
| City |
SHEFFIELD |
| State/province |
South Yorkshire |
| ZIP/Postal code |
S102HQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL23159 |
| Series (1) |
| GSE186798 |
Transcriptomic Profiling Reveals Discrete Poststroke Dementia Neuronal and Gliovascular Signatures |
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