Local Nucleic Acid Analysis of Adherent Cells

Kashyap, Aditya; Huber, Deborah; Autebert, Julien; Kaigala, Govind V.

doi:10.1002/9783527696789.ch7

Cited by 1 publication

(1 citation statement)

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“…24 In addition to providing improved RNA recoveries relative to traditional approaches, microfluidic probes have been designed and applied for the spatially resolved extraction of RNA from layers of adherent cells. 25,26 However, the primary means of RNA detection following the aforementioned microextraction methods involves RT-PCR amplification, precluding detection of the vast majority of RNA modifications. Given the emerging roles of RNA modifications in cell-cell signaling, 27 cell differentiation, 28 and response to changes in chemical microenvironments, 29 a method that combines the advantages of spatially-resolved RNA microextraction with a detection platform that is capable of simultaneously profiling multiple RNA modifications is highly desirable.…”

Section: Introductionmentioning

confidence: 99%

Biphasic Liquid Microjunction Extraction for Profiling Neuronal RNA Modifications by Liquid Chromatography–Tandem Mass Spectrometry

et al. 2020

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RNA modifications are emerging as critical players in the spatiotemporal regulation of gene expression. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables the simultaneous quantification of numerous enzymatically modified RNAs in a biological sample, conventional RNA extraction and enzymatic digestion protocols that are employed prior to analysis have precluded the application of this technique for small-volume samples. In this study, a biphasic liquid microjunction (LMJ) extraction system using coaxial capillaries that direct and aspirate extraction solvents onto a ~350 μm-diameter sample spot was developed and applied for the extraction of RNA from individual cell clusters in the central nervous system of the marine mollusk Aplysia californica. To maximize RNA recoveries, optimized extraction solvents consisting of 10% methanol and chloroform were evaluated under dynamic and static extraction conditions. An MS-compatible RNA digestion buffer was developed to minimize the number of sample transfer steps and facilitate the direct enzymatic digestion of extracted RNA within the sample collection tube. Compared to RNA extraction using a conventional phenol-chloroform method, the LMJ-based method provided 3-fold greater coverage of the neuronal epitranscriptome for similar amounts of tissue and also produced mRNA of sufficient purity for reverse transcription polymerase chain reaction amplification. Using this approach, the expression of RNA-modifying enzymes in a given neuronal cell cluster can be characterized and simultaneously correlated with the LC-MS/MS analysis of RNA modifications within the same subset of neurons.

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Section: Introductionmentioning

confidence: 99%