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mRNA Interactome Capture from Plant Protoplasts
Published on: July 28, 2017
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Multiplex single-cell quantification of rare RNA transcripts from protoplasts in a model plant system
Ulhas S Kadam1,2,3,4, Burkhard Schulz4, Joseph M K Irudayaraj2,3
1VD College of Agricultural Biotechnology (VNMKV), Latur, Maharashtra, 413512, India.
The Plant Journal : for Cell and Molecular Biology
|March 17, 2017
Summary
Researchers developed a new method using gold nanoprobes and surface-enhanced Raman spectroscopy (SERS) for simultaneous detection of rare RNA species in single plant cells. This technique enables precise tracking of intracellular RNA copies, including alternatively spliced variants and microRNAs.
Area of Science:
- Plant molecular biology
- Spectroscopy
- Nanotechnology
Background:
- Studying intracellular RNA in plants at single-cell resolution is challenging.
- Rare RNA species, such as microRNAs and alternatively spliced variants, play crucial roles in gene regulation.
- Existing methods often lack the sensitivity or specificity for simultaneous detection of multiple rare RNAs within single cells.
Purpose of the Study:
- To develop and validate a novel nanoparticle-based Raman spectroscopic sensor for multiplex and simultaneous detection of rare RNA species in single plant cells.
- To demonstrate the applicability of this sensor for intracellular RNA quantification and tracking.
- To assess the potential of this method for analyzing alternatively spliced variants and microRNAs in Arabidopsis thaliana.
Main Methods:
- Utilized gold nanoparticles functionalized with Raman probes and specific nucleic acid probes for targeted RNA detection.
- Employed surface-enhanced Raman spectroscopy (SERS) for high-sensitivity signal detection.
- Confirmed gold nanoprobes' uptake and localization in plant protoplasts using transmission electron microscopy, ICP-MS, and fluorescence imaging.
- Performed multiplex analysis of four specific RNA species (AtPTB2, AtDCL2, miR156a, miR172a) based on distinct spectral features.
Main Results:
- Successfully demonstrated the uptake of functionalized gold nanoprobes by Arabidopsis thaliana protoplasts.
- Achieved multiplex and simultaneous detection of four rare RNA species at single-cell resolution using SERS.
- Validated the accuracy of the SERS-based RNA quantification through in vitro studies.
- Confirmed the low nanotoxicity of the gold probes, ensuring cell viability for analysis.
Conclusions:
- Developed a highly sensitive and accurate SERS-based approach for tracking rare RNAs within plant protoplasts.
- The gold nanosensor platform enables precise quantification of alternatively spliced variants and rare microRNAs in single plant cells.
- This method offers a powerful tool for advancing single-cell studies in plant biology, particularly for analyzing complex RNA dynamics.
