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A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples
Vladimir Vincek1, Mehdi Nassiri, Mehrdad Nadji
1Department of Pathology, Jackson Memorial Medical Center, University of Miami, Miami, Florida, USA. vvincek@med.miami.edu
Abstract:
Preservation of macromolecules (DNA, RNA, and proteins) in tissue is traditionally achieved by immediate freezing of the sample. Although isolation of PCR-able RNA has been reported from formalin-fixed, paraffin-embedded tissues, the process has not been shown to be reproducible because high molecular weight RNA is usually degraded. We investigated the potential value of a new universal molecular fixative (UMFIX, Sakura Finetek USA, Inc., Torrance, California) in preservation of macromolecules in paraffin-embedded tissue. Mouse and human tissues were fixed in UMFIX from 1 hour to 8 weeks. They were then processed by a rapid tissue processing (RTP) system, embedded in paraffin, and evaluated for routine histology as well as for the quality and quantity of DNA, RNA, and proteins. Formalin-fixed tissues were processed by RTP and evaluated in a similar manner. Fresh-frozen samples were used as controls. The morphology of UMFIX-exposed tissue was comparable to that fixed in formalin. High molecular weight RNA was preserved in tissue that was immediately fixed in UMFIX and stored from 1 hour to 8 weeks at room temperature. There were no significant differences between UMFIX-exposed and frozen tissues on PCR, RT-PCR, real-time PCR, and expression microarrays. Similarly, physical and antigenic preservation of proteins in UMFIX tissue was similar to fresh state. Both RNA and proteins were substantially degraded in formalin-fixed and similarly processed specimens. We concluded that it is now possible to preserve histomorphology and intact macromolecules in the same archival paraffin-embedded tissue through the use of a novel fixative and a rapid processing system.
Insights
A new universal molecular fixative (UMFIX) effectively preserves high-molecular-weight RNA and proteins in paraffin-embedded tissues, enabling reproducible molecular analysis alongside histology. This overcomes limitations of traditional formalin fixation for molecular studies.
Area of Science:
- Biotechnology
- Histology
- Molecular Biology
Background:
- Traditional tissue preservation methods like immediate freezing are ideal for macromolecules but impractical for archiving.
- Formalin fixation allows for histological archiving but often degrades nucleic acids and proteins, limiting downstream molecular analysis.
- Existing methods for preserving RNA in formalin-fixed, paraffin-embedded tissues lack reproducibility due to high-molecular-weight RNA degradation.
Purpose of the Study:
- To evaluate a novel universal molecular fixative (UMFIX) for preserving macromolecules (DNA, RNA, proteins) in paraffin-embedded tissues.
- To assess the compatibility of UMFIX with routine histology and rapid tissue processing (RTP) for molecular analysis.
- To compare the preservation quality of UMFIX with traditional formalin fixation and fresh-frozen samples.
Main Methods:
- Mouse and human tissues were fixed using UMFIX for durations ranging from 1 hour to 8 weeks.
- Tissues underwent rapid tissue processing (RTP) and paraffin embedding.
- Histomorphology, DNA, RNA, and protein quality/quantity were assessed using histology, PCR, RT-PCR, real-time PCR, expression microarrays, and antigenicity tests.
- Formalin-fixed tissues and fresh-frozen samples served as comparative controls.
Main Results:
- UMFIX preserved excellent histomorphology, comparable to formalin fixation.
- High-molecular-weight RNA was successfully preserved in UMFIX-treated tissues stored for up to 8 weeks at room temperature.
- Molecular analyses (PCR, RT-PCR, real-time PCR, microarrays) showed no significant differences between UMFIX-treated and fresh-frozen samples.
- Protein preservation in UMFIX-treated tissues was comparable to fresh samples, while formalin fixation led to substantial degradation of both RNA and proteins.
Conclusions:
- UMFIX, in conjunction with RTP, enables the simultaneous preservation of histomorphology and intact macromolecules in archival paraffin-embedded tissues.
- This novel approach overcomes the limitations of formalin fixation, offering a reproducible method for molecular analysis from archived samples.
- UMFIX represents a significant advancement for molecular pathology and biobanking, facilitating integrated diagnostic and research applications.
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