Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Dec 26, 2021; 13(12): 1918-1927
Published online Dec 26, 2021. doi: 10.4252/wjsc.v13.i12.1918
SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells
Andrea Diana, Maria Dolores Setzu, Zaal Kokaia, Roxana Nat, Cristina Maxia, Daniela Murtas
Andrea Diana, Maria Dolores Setzu, Cristina Maxia, Daniela Murtas, Department of Biomedical Sciences, University of Cagliari, Monserrato 09042, Cagliari, Italy
Zaal Kokaia, Laboratory of Stem Cells & Restorative Neurology, Lund Stem Cell Center, Lund University, Lund SE-221 84, Lund, Sweden
Roxana Nat, Institute of Neuroscience, Medical University of Innsbruck, Innsbruck 6020, Austria
Author contributions: Maxia C and Murtas D share senior authorship; Diana A, Setzu MD, Maxia C, and Murtas D contributed to the conception and design of the study, data interpretation, and funding acquisition; Diana A, Kokaia Z, and Nat R contributed to methodology and data acquisition and analysis; Diana A wrote the original draft of the article; Diana A, Maxia C and Murtas D wrote, reviewed and edited the paper, and contributed to project administration and supervision; all authors read and approved the final version of the manuscript.
Supported by the "Fondo Integrativo per la Ricerca" (FIR) of the University of Cagliari, Italy.
Institutional review board statement: The study was reviewed and approved by the Lund/Malmö Ethical Committee of the Lund University, Sweden (ethical permit number No. Dnr 6.1.8-2887/2017).
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Andrea Diana, PhD, Assistant Professor, Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria di Monserrato SS 554 Bivio per Sestu, Monserrato 09042, Cagliari, Italy. diana@unica.it
Received: June 18, 2021
Peer-review started: June 18, 2021
First decision: July 17, 2021
Revised: August 11, 2021
Accepted: December 10, 2021
Article in press: December 10, 2021
Published online: December 26, 2021
ARTICLE HIGHLIGHTS
Research background

Although mRNA analysis is still conventionally achieved by fluorescence in situ hybridization and qRT-PCR, there is a strong need for real-time monitoring of specific RNA transcripts in living cells, both for a qualitative and quantitative assessment. Within this context, SmartFlareTM technology is a reliable tool for evaluating the presence and the upregulation/downregulation of mRNAs in individual living cells. In addition, this nanotechnology offers the advantages of retaining cell viability, morpho-functional and biochemical properties and allowing downstream experiments.

Research motivation

SmartFlareTM technology is a devoted and straightforward method for the spatiotemporal investigation of the in situ mRNA expression in living cells.

Research objectives

To study the dynamics of differentiation-related RNA transcripts in human neural stem cells.

Research methods

The presence of CD133 and OCT4 mRNA-linked nanoprobes in neurosphere-derived cells (from 3 to 30 DIV) was investigated by SmartFlareTM as a reliable insight into neural stem cell differentiation.

Research results

Until 7 DIV, all the cells displayed a strong SmartFlareTM fluorescent signal indicative of CD133 and OCT4 mRNA expression, as single dots encompassing both the cytoplasmic domain and the related processes. Upon 15 DIV, cells showed a marked decrease in the fluorescence, both for CD133 and Oct4 probes. In cells grown for 30 DIV, the CD133 and Oct4 probe uptake was very scant but still consisted of single dot-like elements, representative of a downregulation of the same genes.

Research conclusions

Our findings propose the SmartFlareTM technology as a reliable and straightforward tool in the context of a qualitative expression analysis applied to a broad panel of mRNAs in single living stem cells.

Research perspectives

The NanoFlare technology, such as SmartFlareTM, could enhance the scenario of biomedical applications in the field of marker identification mirroring both normal and pathological conditions, with the advantage of ensuring the physiological integrity of cellular systems.