Multiplex single‐cell quantification of rare RNA transcripts from protoplasts in a model plant system

• Published in: The Plant journal : for cell and molecular biology Vol. 90; no. 6; pp. 1187 - 1195
• Main Authors: Kadam, Ulhas S., Schulz, Burkhard, Irudayaraj, Joseph M.K.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-06-2017
• Subjects: alternate splicing; Alternative splicing; Alternative Splicing - genetics; Botany; Decoration; DNA probes; Dyes; Electron microscopy; Evaluation; Fluorescence; Genes; Gold; Imaging; In vitro methods and tests; Internalization; Intracellular; Mass spectrometry; Mass spectroscopy; MicroRNAs - genetics; miRNA; mRNA; multiplex detection; Multiplexing; Nanoparticles; Nucleic acids; Plant cells; Plant species; Probes; Protoplasts; Protoplasts - metabolism; Raman spectroscopy; Ribonucleic acid; RNA; RNA, Messenger - genetics; RNA, Plant - genetics; Spectra; Spectroscopic analysis; Spectroscopy; Spectrum Analysis, Raman; technical advance; Tracking; Transmission electron microscopy; Water analysis; miRNA; alternate splicing; mRNA; multiplex detection; Raman spectroscopy; technical advance
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.13537

Summary
Here we demonstrate multiplex and simultaneous detection of four different rare RNA species from plant, Arabidopsis thaliana, using surface‐enhanced Raman spectroscopy (SERS) and gold nanoprobes at single‐cell resolution. We show the applicability of nanoparticle‐based Raman spectroscopic sensor to study intracellular RNA copies. First, we demonstrate that gold‐nanoparticles decorated with Raman probes and carrying specific nucleic acid probe sequences can be uptaken by the protoplasts. We confirm the internalization of gold nanoprobes by transmission electron microscopy, inductively‐coupled plasma‐mass spectrometry and fluorescence imaging. Second, we show the utility of a SERS platform to monitor individual alternatively spliced (AS) variants and miRNA copies within single cells. Finally, the distinctive spectral features of Raman‐active dyes were exploited for multiplex analysis of AtPTB2, AtDCL2, miR156a and miR172a. Furthermore, single‐cell studies were validated by in vitro quantification and evaluation of nanotoxicity of gold probes. Raman tag functionalized gold nanosensors yielded an approach for the tracking of rare RNAs within the protoplasts. The SERS‐based approach for quantification of RNAs has the capability to be a highly sensitive, accurate and discerning method for single‐cell studies including AS variants quantification and rare miRNA detection in specific plant species.
Significance Statement
Multiplex analysis of rare RNA transcripts from plant single cells using surface enhanced Raman spectroscopy and gold nanoprobes.