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| Status |
Public on Mar 21, 2024 |
| Title |
DR-A-6h |
| Sample type |
RNA |
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| Source name |
gastrocnemius muscle
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| Organism |
Mus musculus |
| Characteristics |
strain: CD-1 strain
treatment: 30 ug D. russelli venom
group: DR-6h
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| Treatment protocol |
Groups of mice (CD-1 strain, 18-20 grams) received an intramuscular injection, in the right gastrocnemius, of 30 µg venom. Control mice were injected with 50 µL PBS only. At the time intervals of 1 h, 6 h, 24 h following injection, groups of three mice were sacrificed by CO2 inhalation, and the tissue was excised and added to 10% formalin solution in water.
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| Growth protocol |
Groups of mice (CD-1 strain, 18-20 grams)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Four slices of 10 µm thickness from each selected block were deparaffinized using D-Limonene (Sigma), digested with proteinase K, and isolated using RNeasy® Plus Mini Kit QIAGEN, according to the manufacturer's protocol. The final volume of extracted RNA was 14 µL. RNA concentration and purity were assessed using a NanoDrop instrument. Sample concentration was measured at 260 nm and 280 nm, and the ratio of optical density 260/280 and 260/230 were used to test for protein and phenol contamination respectively.
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| Label |
Barcode probe
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| Label protocol |
A total of 21 blocks of FFPE muscle mouse tissue (Control, B. asper (BA) BA-1h, BA-6h, BA-24h, D. russelii (DR) DR-1h, DR-6h, DR-24h) were used for RNA extraction.
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| Hybridization protocol |
The RNA samples (100 ng) were incubated for 16 h at 65 °C in a hybridization buffer containing the CodeSet (reporter and capture probes). Hybridized samples were processed using the Prep Station using high sensitivity protocol, 3 h per 12-sample cartridge.
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| Scan protocol |
The Prep Station purifies the RNA/probe complexes and places them in a cartridge where they are immobilized and aligned for data collection. Data acquisition was carried out in the NanoString nCounter Digital Analyzer with the ‘Max’ Field of View (FOV) setting to 555 images per sample in a 6 h scan per cartridge.
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| Description |
NanoString gene expression profiling was conducted using the mouse nCounter Fibrosis V2 Panel (NanoString Technologies, Seattle, WA, USA), which contains probes specific to 760 endogenous genes and 10 housekeeping genes
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| Data processing |
NanoString gene expression profiling was conducted using the mouse nCounter Fibrosis V2 Panel (NanoString Technologies, Seattle, WA, USA), which contains probes specific to 760 endogenous genes and 10 housekeeping genes (ACAD9, ARMH3, CNOT10, GUSB, MTMR14, NOL7, NUBBP1, PGK1, PPIA, RPLP0). Data were analyzed using ROSALIND (https://rosalind.bio/), with a hyper-scale cloud developed by ROSALIND, Inc. (San Diego, CA), accessed on March 05, 2023. The quality control of samples was verified using spike-ins of positive and negative probes, and housekeeping expression levels.
Normalization, fold changes, and p-values were calculated using criteria provided by NanoString. ROSALIND follows the nCounter Advanced Analysis protocol of dividing counts within a lane by the geometric mean of the normalizer probes from the same lane. The geNorm algorithm was used to select normalizer probes in the Bioconductor package NormqPCR, removing candidate housekeepers with the least stable expression relative to other candidates.
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| Submission date |
Nov 19, 2023 |
| Last update date |
Mar 21, 2024 |
| Contact name |
Ana Karina de Olliiveirra |
| E-mail(s) |
ankaoliv@gmail.com
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| Phone |
434-924-1753
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| Organization name |
UVA
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| Department |
Department of Pathology
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| Lab |
Spatial Biology Core
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| Street address |
1340 Jefferson Park Avenue
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| City |
Charlottesville |
| State/province |
VA |
| ZIP/Postal code |
22908 |
| Country |
USA |
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| Platform ID |
GPL31258 |
| Series (1) |
| GSE248215 |
A complex pattern of gene expression in tissue affected by viperid snake envenoming: the emerging role of autophagy-related genes |
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| Supplementary data files not provided |
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