The posterior portion of the skin that was removed at the week 17 postirradiation time point was utilized for RNA isolation (n = 5 mice) to evaluate gene expression involved in inflammation and fibrosis. At time of harvest, the central irradiated area of dorsal skin was dissected into submillimeter sections, flash-frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from the tissue using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) and quantitated by the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). RNA quality was estimated by the Agilent RNA 6000 Nano Assay (Agilent Technologies, Santa Clara, CA). RNA was subjected to the NanoString nCounter gene expression assay (XT-CSO-MIP1-12; NanoString Technologies, Seattle, WA) as completed by Genomics Shared Resource-Comprehensive Cancer Center according to the manufacturer’s protocol. Raw data were analyzed by nSolver software (NanoString Technologies) using standard settings and were normalized against the housekeeping genes (Abcf1, Hprt, and Oaz1) and fold changes of selected genes involved in inflammation and fibrosis were calculated.
Ncounter gene expression assay xt cso mip1 12
Manufactured by NanoString
The NanoString nCounter gene expression assay is a lab equipment product that allows for the direct digital detection and counting of up to 800 unique RNA or DNA targets in a single reaction. It utilizes a proprietary molecular barcoding technology to provide a sensitive and highly multiplexed analysis of gene expression levels.
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2 protocols using ncounter gene expression assay xt cso mip1 12
1
Gene Expression Analysis of Irradiated Skin
2
Irradiation-Induced Skin Inflammation and Fibrosis
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The posterior portion of the skin that was removed at the week 17 postirradiation time point was utilized for RNA isolation (n = 5 mice) to evaluate gene expression involved in inflammation and fibrosis. At time of harvest, the central irradiated area of dorsal skin was dissected into submillimeter sections, flash‐frozen in liquid nitrogen, and stored at −80 °C. RNA was extracted from the tissue using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) and quantitated by the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). RNA quality was estimated by the Agilent RNA 6000 Nano Assay (Agilent Technologies, Santa Clara, CA). RNA was subjected to the NanoString nCounter gene expression assay (XT‐CSO‐MIP1‐12; NanoString Technologies, Seattle, WA) as completed by Genomics Shared Resource‐Comprehensive Cancer Center according to the manufacturer's protocol. Raw data were analyzed by nSolver software (NanoString Technologies) using standard settings and were normalized against the housekeeping genes (Abcf1, Hprt, and Oaz1) and fold changes of selected genes involved in inflammation and fibrosis were calculated.
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