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
Abstract
BACKGROUND

One of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells. On this matter, a novel platform called SmartFlareTM has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts. Although fluorescence emission does not account for the spatial mRNA distribution, NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases, such as cancer, neurodegenerative pathologies or embryonic developmental disorders.

AIM

To investigate the potential of SmartFlareTM in determining time-dependent mRNA expression of prominin 1 (CD133) and octamer-binding transcription factor 4 (OCT4) in single living cells through differentiation.

METHODS

Brain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres. For the in vitro differentiation, neurospheres were gently dissociated with Accutase solution. Single cells were resuspended in a basic medium enriched with fetal bovine serum, plated on poly-L-lysine-coated glass coverslips, and grown in a lapse of time from 1 to 4 wk. Live cell mRNA detection was performed using SmartFlareTM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlareTM CD133- and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach, taking into account the fluorescence intensity and the number of labeled cells.

RESULTS

In agreement with previous PCR experiments, a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro (DIV). Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern, also detectable in the contacting neural branches. From 15 to 30 DIV, only few cells showed a scattered fluorescent pattern, in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes.

CONCLUSION

SmartFlareTM appears to be a reliable, easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model, preserving cell biological properties in anticipation of downstream experiments.

Keywords: mRNA detection, SmartFlareTM, NanoFlare, Live staining, Nanotechnology, Neural stem cell genes.

Core Tip: The detection of RNA transcripts in living cells is a challenge in embryonic development and cancer related studies. In the last decade, a straightforward and noninvasive approach has emerged, exploiting the combination of nanotechnology and the physiological behavior of stem cells. Although SmartFlareTM technology is far from providing an unambiguous localization of specific mRNAs, it might help in elucidating the time-dependent dynamics of RNA expression at single-cell level, where results are coherent with those coming from both qRT-PCR and fluorescence in situ hybridization (FISH), the gold standards for mRNA analysis.