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 language | English (US) |
|---|---|
| Pages (from-to) | 1187-1195 |
| Number of pages | 9 |
| Journal | Plant Journal |
| Volume | 90 |
| Issue number | 6 |
| DOIs | |
| State | Published - Jun 2017 |
| Externally published | Yes |
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Keywords
- Raman spectroscopy
- alternate splicing
- mRNA
- miRNA
- multiplex detection
- technical advance
ASJC Scopus subject areas
- Genetics
- Plant Science
- Cell Biology
Cite this
Multiplex single-cell quantification of rare RNA transcripts from protoplasts in a model plant system. / Kadam, Ulhas S.; Schulz, Burkhard; Irudayaraj, Joseph Maria Kumar.
In: Plant Journal, Vol. 90, No. 6, 06.2017, p. 1187-1195.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Multiplex single-cell quantification of rare RNA transcripts from protoplasts in a model plant system
AU - Kadam, Ulhas S.
AU - Schulz, Burkhard
AU - Irudayaraj, Joseph Maria Kumar
PY - 2017/6
Y1 - 2017/6
N2 - 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.
AB - 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.
KW - Raman spectroscopy
KW - alternate splicing
KW - mRNA
KW - miRNA
KW - multiplex detection
KW - technical advance
UR - http://www.scopus.com/inward/record.url?scp=85018350209&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85018350209&partnerID=8YFLogxK
U2 - 10.1111/tpj.13537
DO - 10.1111/tpj.13537
M3 - Article
C2 - 28301688
AN - SCOPUS:85018350209
VL - 90
SP - 1187
EP - 1195
JO - Plant Journal
JF - Plant Journal
SN - 0960-7412
IS - 6
ER -