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. 2020 Sep 15;92(18):12647-12655.
doi: 10.1021/acs.analchem.0c02830. Epub 2020 Aug 27.

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

Kevin D Clark  1 , Marina C Philip  2 , Yanqi Tan  2 , Jonathan V Sweedler  1   2
Affiliations

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

Kevin D Clark et al. Anal Chem. .

Abstract

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 a 3-fold greater coverage of the neuronal epitranscriptome for similar amounts of tissues 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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Figures

Figure 1.
Figure 1.
(A) Schematic of the LMJ RNA extraction setup and analytical workflow. (B) Dried tRNA sample spot and the extraction footprint left by the LMJ probe following extraction. (C) Representative EICs for several modified nucleosides following digestion of tRNA samples spiked with 10% methanol (black trace) or TRIzol (red trace) aqueous extraction solvents. EICs for the positional isomers of methyladenosine and methylcytidine were at m/z 282.12 ± 0.01 and m/z 258.11 ± 0.01, respectively.
Figure 2.
Figure 2.
Comparison of yeast total tRNA digestion using a Tris buffer or MS-compatible ammonium acetate buffer. (A) PAGE of intact yeast total tRNA standard (lane 1), nucleosides standard (lane 2), digestion in Tris buffer for 3 h (lanes 3 and 4), and digestion in ammonium acetate buffer for 3 h (lanes 5 and 6), 2 h (lane 7), and 1 h (lane 8). (B) Overlays of representative EICs for several modified nucleosides obtained for the studied digestion conditions. EICs for positional isomers of methyladenosine (m/z 282.12), methylcytidine (m/z 258.11), and methylguanosine (m/z 298.12), as well as 5-aminomethyl-2-thiouridine (nm5s2U, m/z 290.08) and N2,N2-dimethylguanosine (m2,2G, m/z 312.13), are plotted with a ±0.01 m/z window. Mean peak areas (n = 3) are shown in panel (C) where error bars represent standard deviation. No significant differences (p >0.05/n) were observed for modified nucleoside peak areas under the two conditions studied.
Figure 3.
Figure 3.
Analysis of neuronal RNA modifications from CNS tissue of Aplysia californica. (A) Schematic of the animal’s CNS, along with a photograph of the buccal ganglion. (B) EICs corresponding to the canonical nucleoside cytidine (m/z 244.09 ± 0.01), the modified nucleoside inosine (m/z 269.09 ± 0.01), and positional isomers of methyladenosine (m/z 282.12 ± 0.01) detected using either LMJ RNA extraction or conventional LLE prior to RNA digestion and LC–MS/MS analysis. (C) Representative MS/MS spectrum for 2’-O-methyladenosine (Am) obtained following LMJ-based extraction.
Figure 4.
Figure 4.
(A) Workflow for simultaneous LC–MS detection of modified nucleosides and RT-PCR analysis of mRNA for two RNA methyltransferases from A. californica. Identified B cells from the buccal ganglion are labeled B3–B5. (B) Agarose gel electrophoresis following RT-PCR of two RNA methyltransferases, namely, DNMT2 (lanes denoted with 1) and m7G TMT (lanes denoted with 2). Lanes were further grouped and marked with a letter (a–c) indicating the extraction method used: (a) dynamic LMJ extraction, (b) conventional LLE, and (c) static LMJ extraction. H4 ctrl represents a positive control. (C) EICs for 1-methyladenosine (m1A), inosine (I), and 7-methylguanosine (m7G) were generated from the same static LMJ extraction aliquot used for the results shown in lanes c1 and c2.
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