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

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

doi:10.3390/ijms21207540

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.

Highlights

Clinical samples from surgery and biopsy procedures are fixed for histopathological analysis
We have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA
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

Summary

Introduction

Clinical samples from surgery and biopsy procedures are fixed for histopathological analysis. An optimal morphological preservation was the sole requirement for fixation, whereas nowadays nucleic acids preservation for onco-pathology has become an important requisite for gene expression profiling and sequencing aimed at defining reliable diagnostic and prognostic parameters [1]. In the past Bouin’s solution was applied in certain hospitals for its ability to preserve some morphological details, such as nuclear conformation [2]. It is well-known that in formalin-fixed and Bouin’s fixed samples the quality of nucleic acid is lower than in alcohol-based fixatives [4,5,6] and that formalin fixation has led to the formation of mono-methylol adducts (hydroxymethyl derivatives) in nucleic acids and proteins and cross-links between them [7]. For other fixatives, included Bouin’s solution, their effect in modifying

Objectives

Methods

Results