Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5508105 | Biochimica et Biophysica Acta (BBA) - General Subjects | 2017 | 45 Pages |
Abstract
The recently announced new methodologies to detect mRNA molecules in single cells offer opportunities for research, medicine and molecular diagnostics. The NanoFlare RNA Detection Probes are tools for characterizing RNA content (not localization) using fluorescence-based approaches in living cells. Combined with flow cytometry, NanoFlares have expanded the available possibilities of quantitative analysis of mRNA level in a single cell. Herein we present that in some cases, the specific NanoFlare probes (SmartFlares) detect different amounts of mRNA compared to qPCR. Using the previously published model, in which we studied influence of BCR-ABL oncogene on BRCA1 mRNA translation, we found that the NanoFlare-mediated measurement of mRNA was affected by the assembly of stress granules, structures which store mRNA in complexes with RNA binding proteins. With the usage of chemical compounds we confirmed that under conditions supporting assembly of stress granules, the detection of mRNAs by these probes was decreased, whereas disassembly resulted in the increased mRNAs detection. Altogether, we showed that assembly of stress granules could interfere with mRNA accessibility to the NanoFlare RNA Detection Probes, indicating that the SmartFlares could recognize only the translationally active pool of mRNA, contrary to qPCR. This can significantly influence the quality of obtained data and should be taken into consideration while planning the analysis of mRNA markers using NanoFlares.
Keywords
FBSmRNA detectionATF-4emetineThapsigargineIF2αSGSCMLqPCRmRNAmessenger RNATIARDiagnosticCancerfetal bovine serumendoplasmic reticulumAU-rich elementactivating transcription factor 4Chronic myeloid leukemiaUTR یا untranslated regions untranslated regionAREquantitative polymerase chain reactionBRCA1Stress granules
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Authors
Magdalena Wolczyk, Paulina Podszywalow-Bartnicka, Lukasz Bugajski, Katarzyna Piwocka,