High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells
| Autor: | Helén Nilsson, Martin Johansson, Krzysztof M. Krawczyk |
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| Rok vydání: | 2014 |
| Předmět: |
RNA Stability
Polymers Bioengineering Biology Buffers Sodium Chloride Applied Microbiology and Biotechnology Antibodies Flow cytometry Cell Line NaCl Cell Line Tumor Formaldehyde Gene expression medicine Humans Multiplex Intracellular antibody labeling Fluorescent Dyes medicine.diagnostic_test Staining and Labeling Keratin-8 Methanol Keratin-7 RNA General Medicine Carbocyanines Flow Cytometry Fixation Molecular biology Cell biology Cell culture Cancer and Oncology Fluorescence-activated cell sorting Nucleic acid Intracellular RNA integrity Biotechnology |
| Zdroj: | Journal of Biotechnology; 192(Sep 30), pp 62-65 (2014) |
| ISSN: | 1873-4863 |
| Popis: | Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields. |
| Databáze: | OpenAIRE |
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