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Cytoplasmic RNA in undifferentiated neural stem cells: a potential label-free Raman spectral marker for assessing the undifferentiated status

Ghita, Adrian; Pascut, Flavius C.; Mather, Melissa; Sottile, Virginie; Notingher, Ioan

Authors

Adrian Ghita

Flavius C. Pascut

Melissa Mather

Virginie Sottile

Ioan Notingher

Abstract

Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ~700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (~1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA.

Journal Article Type Article
Publication Date Mar 13, 2012
Journal Analytical Chemistry
Print ISSN 0003-2700
Electronic ISSN 1520-6882
Publisher American Chemical Society
Peer Reviewed Peer Reviewed
Volume 84
Issue 7
Institution Citation Ghita, A., Pascut, F. C., Mather, M., Sottile, V., & Notingher, I. (2012). Cytoplasmic RNA in undifferentiated neural stem cells: a potential label-free Raman spectral marker for assessing the undifferentiated status. Analytical Chemistry, 84(7), doi:10.1021/ac202994e
DOI https://doi.org/10.1021/ac202994e
Publisher URL https://pubs.acs.org/doi/abs/10.1021/ac202994e
Copyright Statement Copyright information regarding this work can be found at the following address: http://eprints.nottingh.../end_user_agreement.pdf

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