Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry
نویسندگان
چکیده
Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation translation central nervous system (CNS). Recent evidence has linked specific neuronal to learning and memory paradigms. Unfortunately, conventional methods for detection these epitranscriptomic features are only capable characterizing highly abundant bulk tissues, precluding assessment unique PTM profiles that may exist individual neurons within activated behavioral circuits. In this protocol, approach is described—single-neuron modification analysis by mass spectrometry (SNRMA-MS)—to simultaneously detect quantify numerous modified ribonucleosides single neurons. The validated using marine mollusk, Aplysia californica, beginning with surgical isolation enzymatic treatment major CNS ganglia expose neuron cell bodies, followed manual single-neuron sharp needles a micropipette. Next, mechanical thermal sample small volume buffer done liberate from subsequent digestion. Modified nucleosides then identified quantified optimized liquid chromatography-mass method. SNRMA-MS employed establish patterns single, A. californica have known morphologies functions. Examples qualitative quantitative presented highlight heterogeneous distribution across networks.
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ژورنال
عنوان ژورنال: Journal of Visualized Experiments
سال: 2022
ISSN: ['1940-087X']
DOI: https://doi.org/10.3791/63940-v