کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8416869 | 1545653 | 2018 | 4 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Isolation of intact RNA from murine CD4+ T cells after intracellular cytokine staining and fluorescence-activated cell sorting
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کلمات کلیدی
FACSICSRNA isolation - جداسازی RNAfluorescence-activated cell sorting - دسته بندی سلول های فعال فلورسنسintracellular cytokine staining - رنگ آمیزی سیتوکین داخل سلولیRIN - رینT cells - سلول های تیRNA Integrity Number - شماره یکپارچه RNATranscriptomics - متن ترانهParaffin-embedded - پارافین جاسازی شده
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوتکنولوژی یا زیستفناوری
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چکیده انگلیسی
Intracellular cytokine staining (ICS) is a powerful method for identifying functionally distinct lymphocyte subsets, and for isolating these by fluorescence activated cell sorting (FACS). Although transcriptomic analysis of cells sorted on the basis of ICS has many potential applications, this is rarely performed because of the difficulty in isolating intact RNA from cells processed using standard fixation and permeabilization buffers for ICS. To address this issue, we compared three buffers shown previously to preserve RNA in nonhematopoietic cells subjected to intracellular staining for their effects on RNA isolated from T lymphocytes processed for ICS. Our results showed that buffers containing the recombinant ribonuclease inhibitor RNasin or high molar concentrations of salt yielded intact RNA from fixed and permeabilized T cells. As proof of principle, we successfully used the buffer containing RNasin to isolate intact RNA from CD4+ T cells that were sorted by FACS on the basis of specific cytokine production, thus demonstrating the potential of this approach for coupling ICS with transcriptomic analysis.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 456, May 2018, Pages 77-80
Journal: Journal of Immunological Methods - Volume 456, May 2018, Pages 77-80
نویسندگان
Shajo Kunnath-Velayudhan, Steven A. Porcelli,