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-1 is 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-1 is 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.

Contribution of Ribonucleic Acid (RNA) to the Fourier Transform Infrared (FTIR) Spectrum of Eukaryotic Cells

ZUCCHIATTI, PAOLO
Writing – Original Draft Preparation
;
MITRI, ELISA
Membro del Collaboration Group
;
BILLÈ, FULVIO
Software
;
VACCARI, LISA
Supervision
2016-01-01

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-1 is 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-1 is 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.
2016
Pubblicato
http://pubs.acs.org/doi/abs/10.1021/acs.analchem.6b02744
File in questo prodotto:
File Dimensione Formato  
acs%2Eanalchem%2E6b02744.pdf

Accesso chiuso

Tipologia: Documento in Versione Editoriale
Licenza: Copyright Editore
Dimensione 1.72 MB
Formato Adobe PDF
1.72 MB Adobe PDF   Visualizza/Apri   Richiedi una copia
ac6b02744_si_001.pdf

Accesso chiuso

Descrizione: supporting information
Tipologia: Altro materiale allegato
Licenza: Copyright Editore
Dimensione 815.38 kB
Formato Adobe PDF
815.38 kB Adobe PDF   Visualizza/Apri   Richiedi una copia
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2931740
Citazioni
  • ???jsp.display-item.citation.pmc??? 12
  • Scopus 43
  • ???jsp.display-item.citation.isi??? 44
social impact