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

Ulhas S. Kadam, Burkhard Schulz, Joseph M.K. Irudayaraj

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1187-1195
Number of pages9
JournalPlant Journal
Volume90
Issue number6
DOIs
StatePublished - Jun 2017
Externally publishedYes

Keywords

  • Raman spectroscopy
  • alternate splicing
  • mRNA
  • miRNA
  • multiplex detection
  • technical advance

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

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