1. A Novel HPLC-Based Method to Investigate on RNA after Fixation
- Author
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Paolo Fattorini, Cristina Forzato, Eros Azzalini, Eleonora De Martino, Serena Bonin, Vincenzo Canzonieri, Domenico Tierno, Giorgio Stanta, Fattorini, P., Forzato, C., Tierno, D., De Martino, E., Azzalini, E., Canzonieri, V., Stanta, G., and Bonin, S.
- Subjects
Tissue Fixation ,High-performance liquid chromatography ,lcsh:Chemistry ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Hydroxymethyl ,Nucleotide ,lcsh:QH301-705.5 ,Spectroscopy ,Fixation (histology) ,degradation ,chemistry.chemical_classification ,0303 health sciences ,modification ,biology ,fixation ,General Medicine ,3. Good health ,Computer Science Applications ,Liver ,nuclease digestion ,030220 oncology & carcinogenesis ,Catalysis ,Article ,Inorganic Chemistry ,RNA ,nucleotide-monophosphate ,HPLC ,hm-NMP ,ISO standards ,03 medical and health sciences ,Fixatives ,Animals ,Physical and Theoretical Chemistry ,Molecular Biology ,030304 developmental biology ,Nuclease ,Chromatography ,Tissue Embedding ,Organic Chemistry ,Chemical modification ,chemistry ,lcsh:Biology (General) ,lcsh:QD1-999 ,Nucleic acid ,biology.protein ,Chromatography, Liquid - 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.
- Published
- 2020