A Novel HPLC-Based Method to Investigate on RNA after Fixation

• Main Authors: Paolo Fattorini, Cristina Forzato, Domenico Tierno, Eleonora De Martino, Eros Azzalini, Vincenzo Canzonieri, Giorgio Stanta, Serena Bonin
• Format: Article
• Language: English
• Published: MDPI AG 2020-10-01
• Series: International Journal of Molecular Sciences
• Subjects: RNA; fixation; nuclease digestion; nucleotide-monophosphate; HPLC; degradation
• Online Access: https://www.mdpi.com/1422-0067/21/20/7540
• ISSN: 1661-6596; 1422-0067

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.