SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells

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 SmartFlare^TM 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 SmartFlare^TM 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 SmartFlare^TM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlare^TM 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

SmartFlare^TM 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.

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 SmartFlare^TM 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.

• Citation: Diana A, Setzu MD, Kokaia Z, Nat R, Maxia C, Murtas D. SmartFlare^TM is a reliable method for assessing mRNA expression in single neural stem cells. World J Stem Cells 2021; 13(12): 1918-1927
• URL: https://www.wjgnet.com/1948-0210/full/v13/i12/1918.htm
• DOI: https://dx.doi.org/10.4252/wjsc.v13.i12.1918