Non-formalin fixative versus formalin-fixed tissue: A comparison of histology and RNA quality

Abstract

Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin. Histology of all formalin-fixed and PAXgene fixed samples was comparable. RNA with acceptable integrity scores could be isolated from all embedded tissues, 4.0 to 7.2 for formalin and 6.4 to 7.7 for PAXgene, as compared to 8.0 to 9.2 for fresh frozen samples. While RNA with acceptable RINs (RNA integrity number) could be isolated from formalin-fixed samples, in microcapillary electrophoresis this RNA separated with a slower migration rate and displayed diffuse, less focused peaks for ribosomal RNA as compared to RNA from frozen or PAXgene fixed samples. Furthermore, RNA from formalin-fixed tissues exhibited inhibition in quantitative RT PCR assays which increased with increasing amplicon length, while RNA from PAXgene fixed samples did not show such inhibition. In conclusion, our results demonstrate that excluding other preanalytical factors, PAXgene Tissue System preserves histology similarly to formalin, but unlike formalin, does not chemically modify RNA. RNA purified from PAXgene fixed tissues is of high integrity and performs as well as RNA from fresh frozen tissue in RT PCR regardless of amplicon length.

Comprehensive DNA Methylation and Extensive Mutation Analyses of HER2-Positive Breast Cancer

“…The PAXgene Tissue System is a recently developed tissue stabilization technology. So far, researchers have reported that RNA and DNA purified from PAXgene-fixed tissues are of high integrity and that they perform as well as those from fresh frozen tissue [15,16,17]. …”

When Tissue Antigens and Antibodies Get Along

“…21 For molecular analysis, PAXgene preserves RNA better than does formalin. 130 The immunoreactivity of antigens in fixed and paraffinembedded tissue varies markedly depending on the antigen and more specifically on the epitope recognized by the antibody. 8,59 In a comparison of the effect of different fixatives (strong cross-linkers, weak cross-linkers, coagulant, and combination coagulant/cross-linkers) on antigen immunoreactivity in mouse tissue, formaldehyde, followed by the combination fixatives, performed better than the other types.…”

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.…”