SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells
Diana, Andrea ; Setzu, Maria Dolores ; Kokaia, Zaal LU
; Nat, Roxana
; Maxia, Cristina
and Murtas, Daniela
(2021)
In World Journal of Stem Cells
13(12).
p.1918-1927
- 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... (More)
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.
(Less)
- author
- Diana, Andrea
; Setzu, Maria Dolores
; Kokaia, Zaal
LU
; Nat, Roxana
; Maxia, Cristina
and Murtas, Daniela
- organization
- publishing date
- 2021
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Live staining, Mrna detection, Nanoflare, Nanotechnology, Neural stem cell genes, Smartflaretm
- in
- World Journal of Stem Cells
- volume
- 13
- issue
- 12
- pages
- 10 pages
- publisher
- Baishideng Publishing Group
- external identifiers
-
- pmid:35069990
- scopus:85122562525
- ISSN
- 1948-0210
- DOI
- 10.4252/wjsc.v13.i12.1918
- language
- English
- LU publication?
- yes
- id
- 7c808343-6e6d-44f1-a0fb-9bd6f3a44639
- date added to LUP
- 2022-03-07 14:53:34
- date last changed
- 2024-03-29 05:39:13
@article{7c808343-6e6d-44f1-a0fb-9bd6f3a44639,
abstract = {{<p>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.</p>}},
author = {{Diana, Andrea and Setzu, Maria Dolores and Kokaia, Zaal and Nat, Roxana and Maxia, Cristina and Murtas, Daniela}},
issn = {{1948-0210}},
keywords = {{Live staining; Mrna detection; Nanoflare; Nanotechnology; Neural stem cell genes; Smartflaretm}},
language = {{eng}},
number = {{12}},
pages = {{1918--1927}},
publisher = {{Baishideng Publishing Group}},
series = {{World Journal of Stem Cells}},
title = {{SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells}},
url = {{http://dx.doi.org/10.4252/wjsc.v13.i12.1918}},
doi = {{10.4252/wjsc.v13.i12.1918}},
volume = {{13}},
year = {{2021}},
}