Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry.
J Vis Exp
; (182)2022 04 21.
Article
en En
| MEDLINE
| ID: mdl-35532275
ABSTRACT
Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described-single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)-to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Aplysia
/
ARN
Tipo de estudio:
Guideline
/
Qualitative_research
Límite:
Animals
Idioma:
En
Revista:
J Vis Exp
Año:
2022
Tipo del documento:
Article
Pais de publicación:
EEUU
/
ESTADOS UNIDOS
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ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
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UNITED STATES OF AMERICA
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US
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USA