A novel hplc-based method to investigate on rna after fixation

Paolo Fattorini, Cristina Forzato, Domenico Tierno, Eleonora De Martino, Eros Azzalini, Vincenzo Canzonieri, Giorgio Stanta, Serena Bonin

Research output: Contribution to journalArticlepeer-review

Abstract

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

Original languageEnglish
Article number7540
Pages (from-to)1-22
Number of pages22
JournalInternational Journal of Molecular Sciences
Volume21
Issue number20
DOIs
Publication statusPublished - Oct 2 2020

Keywords

  • Degradation
  • Fixation
  • Hm-NMP
  • HPLC
  • ISO standards
  • Modification
  • Nuclease digestion
  • Nucleotide-monophosphate
  • RNA

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

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