دورية أكاديمية
Quantification of mRNA translation in live cells using single-molecule imaging.
| العنوان: | Quantification of mRNA translation in live cells using single-molecule imaging. |
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| المؤلفون: | Khuperkar D; Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands., Hoek TA; Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands., Sonneveld S; Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands., Verhagen BMP; Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands., Boersma S; Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands., Tanenbaum ME; Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands. m.tanenbaum@hubrecht.eu. |
| المصدر: | Nature protocols [Nat Protoc] 2020 Apr; Vol. 15 (4), pp. 1371-1398. Date of Electronic Publication: 2020 Feb 19. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Nature Pub. Group Country of Publication: England NLM ID: 101284307 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1750-2799 (Electronic) Linking ISSN: 17502799 NLM ISO Abbreviation: Nat Protoc Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: London, UK : Nature Pub. Group, 2006- |
| مواضيع طبية MeSH: | Image Processing, Computer-Assisted/*methods , Protein Biosynthesis/*genetics , RNA, Messenger/*analysis , Single Molecule Imaging/*methods, HEK293 Cells ; Humans ; RNA, Messenger/genetics |
| مستخلص: | mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3'-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2-4 d to complete (after cell lines expressing all required transgenes have been generated). |
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| المشرفين على المادة: | 0 (RNA, Messenger) |
| تواريخ الأحداث: | Date Created: 20200221 Date Completed: 20200417 Latest Revision: 20210218 |
| رمز التحديث: | 20240628 |
| DOI: | 10.1038/s41596-019-0284-x |
| PMID: | 32076351 |
| قاعدة البيانات: | MEDLINE |
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Full Text Finder | تدمد: | 1750-2799 |
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| DOI: | 10.1038/s41596-019-0284-x |