Raman spectroscopy for DNA quantification in cell nucleus

Abstract

Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm 21. When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate. V C 2014 International Society for Advancement of Cytometry

Monitoring the biochemical changes occurring to human keratinocytes exposed to solar radiation by Raman spectroscopy

“…To gain further insight into the deleterious effects of SSR in HaCaT cells, Raman spectroscopy was used to characterize and identify biochemical signatures within the nucleus of the cells, immediately after and 24 hours after irradiation [11, 20, 21]. Figure 3 presents the average Raman spectrum of a HaCaT cell, highlighting the characteristic bands related to nucleic acids, which confirm the Raman spectra were acquired in the nucleus and not in another subsection of the cell [59, 60]. The comparison of the average spectrum of control and irradiated cells (immediately and 24 hours post‐exposure), respectively do not clearly elucidate DNA damage in cells after irradiation (Figure S1).…”

Quality assessment of cryopreserved biospecimens reveals presence of intact biomolecules

“…Acquisition of spectra of randomly chosen 19 samples each of short‐term (OS 2T, OS 4N, OS 6T, OS 8T, OS 10N, OS 10T, OS 12T, OS 13T, BS 1N, BS 2N, BS 3N, BS 7N, BS 7T, BS 9N, CS 1T, CS 3N, CS 6N, CS 6T, CS 10T) and long‐term (OL 3N, OL 8N, OL 10N, OL 10T, BL 1N, BL 1T, BL 2N, BL 2T, BL 3T, BL 5T, BL 6N, BL 6T, BL 8N, CL 2N, CL 2T, CL 6N, CL 6T, CL 9N, CL 9T) cryopreserved genomic DNA showed prominent peaks previously reported in human genomic DNA; 780 cm −1 —cytosine, thymine, 915 cm −1 —deoxyribose ring, 1061 cm −1 —d( v CO), 1251 cm −1 —cytosine, adenine, 1345 cm −1 —adenine, guanine, 1468 cm −1 —d(CH2 δ ), 1576 cm −1 —ring mode guanine, adenine vibration (Figure A,B). The absence of characteristic RNA, amide I and amide III bands at around 815 cm −1 , 1300 cm −1 , 1655 cm −1 , respectively, confirms the samples are devoid of RNA and protein , . Notably, we observed a distinct sharp band at 1012 cm −1 in all the extracted genomic DNA samples irrespective of the cryopreservation period which instigated the need to analyze other components of the PAXgene Tissue DNA Kit (PreAnalytiX).…”

Resonance Raman spectra for the in-situ identification of bacteria strains and their inactivation mechanism

“…However, these scattering data provide very little information, concerning bacterial identity and in addition do not differentiate between live and dead bacteria and neither provide information that may be used to identify the bacterial strain. In contrast, Raman spectroscopy provides incisive information, which allows us to assign bands to specific bacterial component bonds (12)(13)(14)(15)(16)(17), therefore, protein compounds such as Tryptophan, Tyrosine and metalloporphyrins, in solution have been extensively studied by Raman spectroscopy (18)(19)(20). Raman spectroscopy, however, has the disadvantage of requiring higher concentration of bacteria for accurate assignment of several relevant (protein, pigments, DNA) weak vibrational bands.…”