Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives

Abstract

Formalin fixation preserves tissue morphology at the expense of macromolecule integrity. Freshly frozen samples are the golden standard for DNA and RNA analyses but require laborious deep-freezing and frozen sectioning for morphological studies. Alternative tissue stabilisation methods are therefore needed. We analysed the preservation of nucleic acids, immunohistochemical staining properties and tissue morphology in paraffin-embedded clinical tissue samples fixed with Z7, RCL2, PAXgene, Allprotect and RNAlater. Formalin-fixed and deep-frozen samples were used as controls. Immunohistochemical analyses showed good preservation of antigenicity in all except Allprotect and RNAlater-fixed samples. RNA quality, based on RNA integrity number value by Bioanalyzer, was comparable with freshly frozen samples only in PAXgene-fixed samples. According to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, RNA from PAXgene samples yielded results similar to freshly frozen samples. No difference between fixatives was seen in DNA analyses (PCR and real-time PCR). In conclusion, PAXgene seems to be superior to other molecular fixatives and formaldehyde.

Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system

“…Clinical sequencing of meningioma paraffin-embedded tissues owing to the high quality of DNA extracted from PAXgene-fixed paraffinembedded specimens 44,45 and conserved histological morphology. 46 In fact, we analyzed two cases of meningioma using genomic DNA extracted from formalin-fixed paraffin-embedded tissue and obtained similar but imprecise results of copynumber alterations (data not shown), mostly because of DNA fragmentation by formalin fixation.…”

Laser microdissection of semi-thin sections from plastic-embedded tissue for studying plant–organism developmental processes at single-cell resolution

“…In LM procedures using paraffin as a tissue support, the crystalline nature of the paraffin interferes with the visualization of the cells due to the refraction of the light as it passes through the paraffin. Furthermore, loss of biological molecules in the form of RNA happens as the paraffin is dissolved away (Jonsson & Lagerstedt 1958;Nair 1958;Groelz et al 2013;Staff et al 2013). As can be imagined, this situation may be particularly problematic for small RNA species like microRNAs (miRNAs) that would be easily liberated from the section.…”

“…However, for studies focused on gene expression, the paraffin-embedding procedure has drawbacks. The major drawback of paraffin is contamination with RNAses, resulting in RNA degradation (Jonsson & Lagerstedt 1957;Nair 1958;Groelz et al 2013;Staff et al 2013). Furthermore, some of the early steps in the paraffin embedding procedure, prior to the casting of tissue into molds, involve temperatures >50°C which has negative impacts on the tissue.…”

The concentration of ethanol affects its penetration rate in bovine cardiac and hepatic tissues

“…Ethanol is a frequently used tissue fixative [2,3]. The combination of ethanol with other fixatives has advantage to preserve the integrity of nucleic acids [2,[4][5][6]12] while improving the visibility of antigens [6].…”

“…Ethanol is a commonly used fixative and it can be used alone [2,3] or in combination with other fixatives [2,[4][5][6]. Fixation of the inner layers of tissues depends on the ability of the fixative to diffuse into the tissues.…”