title: Contribution of Ribonucleic Acid (RNA) to the Fourier Transform Infrared (FTIR) Spectrum of Eukaryotic Cells.
abstract: We report on an optimized protocol for the digestion of cellular RNA, which minimally affects the cell membrane integrity, maintaining substantially unaltered the vibrational contributions of the other cellular macromolecules. The design of this protocol allowed us to collect the first Fourier transform infrared (FTIR) spectra of intact hydrated B16 mouse melanoma cells deprived of RNA and to highlight the in-cell diagnostic spectral features of it. Complementing the cellular results with the FTIR analysis of extracted RNA, ds-DNA, ss-cDNA and isolated nuclei, we verified that the spectral component centered at ∼1220 cm-1is a good qualitative and semiquantitative marker of cellular DNA, since it is minimally affected by cellular RNA removal. Conversely, the band centered at ∼1240 cm-1, conventionally attributed to RNA, is only a qualitative marker of it, since its intensity is majorly influenced by other macromolecules containing diverse phosphate groups, such as phospholipids and phosphorylated proteins. On the other hand, we proved that the spectral contribution centered at ∼1120 cm-1is the most reliable indicator of variations in cellular RNA levels, that better correlates with cellular metabolic activity. The achievement of these results have been made possible also by the implementation of new methods for baseline correction and automated peak fitting, presented in this paper.
authors.affiliation: Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5, 34151, Trieste, Italy.|Dipartimento di Fisica, Università degli Studi di Trieste , via Valerio 2, 34127 Trieste, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Zucchiatti
authors.affiliation: Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5, 34151, Trieste, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Mitri
authors.affiliation: Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5, 34151, Trieste, Italy.
authors.forename: Saša
authors.initials: S
authors.lastname: Kenig
authors.affiliation: Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5, 34151, Trieste, Italy.
authors.forename: Fulvio
authors.initials: F
authors.lastname: Billè
authors.affiliation: Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5, 34151, Trieste, Italy.
authors.forename: George
authors.initials: G
authors.lastname: Kourousias
authors.affiliation: Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5, 34151, Trieste, Italy.
authors.forename: Diana Eva
authors.initials: DE
authors.lastname: Bedolla
authors.affiliation: Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5, 34151, Trieste, Italy.
authors.forename: Lisa
authors.initials: L
authors.lastname: Vaccari
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D017830:Octoxynol / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D025341:Principal Component Analysis; D012334:RNA, Neoplasm / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D004277:DNA, Single-Stranded; D010743:Phospholipids; D010748:Phosphopeptides; D012334:RNA, Neoplasm; D017830:Octoxynol; D004247:DNA; D012260:Ribonucleases