Optimization of RNA extraction protocol for long-term archived formalin-fixed paraffin-embedded tissues of horses.
Abstract: A suitable RNA extraction protocol was established to gain high quality RNA from formalin-fixed paraffin-embedded tissues to perform reliable molecular assays either applicable for using FFPE tissue archives or tissues with harsh formalin-fixation. Eighteen FFPE samples from the central nervous system of horses, stored up to 11 years, were used as archive cases. To test the influence of the fixation period, brain, liver, kidney, and skeletal muscle tissue fragments from another horse, were treated either with water or tris-acetate-EDTA buffer after fixation under different timepoints with 10% unbuffered formalin. Two deparaffinization methods and three proteinase K-based lysis step were tested and translated into three protocols. After detailed statistical analysis it was determined that a longer period and increase in volume of proteinase K incubation provide higher yields and purity of RNA (P < 0.01) of archived samples. Alongside, amplification of equid-housekeeping gene up to 298 bp was successful with the protocol adaptations. For different formalin-fixation timepoints, it was demonstrated that the right choice for treatment and formalin-fixation period is organ-related (P ≤ 0.05). Essentially, little alterations to pre-existing extraction protocols unwound the RNA of up to 11-year-old samples, enabling the use of FFPE tissue archives or e.g. harshly fixed material needed in infection research under high biosafety levels for a variety of molecular analysis.
Copyright © 2019 Elsevier Inc. All rights reserved.
Publication Date: 2019-07-23 PubMed ID: 31348903DOI: 10.1016/j.yexmp.2019.104289Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study focuses on improving the extraction process of RNA from long-term, preserved tissue samples (formalin-fixed paraffin-embedded, or FFPE) of horses. The research finds that adjustments to existing protocols can enhance both the yield and purity of the extracted RNA, enabling researchers to use older tissue samples in a range of molecular analyses.
Objective of the Research
- The primary aim of this research was to optimize the protocol for RNA extraction from long-term archived formalin-fixed paraffin-embedded (FFPE) tissues of horses. This RNA can then be used to perform reliable molecular assays either with FFPE tissue archives or with tissues subjected to harsh formalin-fixation.
About the Test Samples
- The test samples used in this experiment included 18 FFPE samples from the central nervous system of horses, which had been stored for up to 11 years.
- To understand the influence of the fixation period on the efficacy of the extraction protocol, tissue fragments from the brain, liver, kidney, and skeletal muscle of another horse were either treated with water or tris-acetate-EDTA buffer after fixation at different timepoints with 10% unbuffered formalin.
Methodology and Protocols
- The experiments involved testing two deparaffinization methods and three protocols that are based on proteinase K-based lysis steps.
- Following statistical analysis, it was determined that increasing the period and volume of proteinase K incubation resulted in higher yields and purity of RNA from the archived samples.
- The research also demonstrated the success of amplifying the equid-housekeeping gene up to 298 base pairs using the adapted protocols.
Findings of the Study
- It was identified that the proper choice of treatment and the formalin-fixation period is organ-specific.
- Using modifications of existing protocols led to the extraction of RNA from up to 11-year-old samples.
- This suggests the potential of using FFPE tissue archives or even harshly fixed materials in infection research at high biosafety levels for an array of molecular analyses.
Cite This Article
APA
Boos GS, Nobach D, Failing K, Eickmann M, Herden C.
(2019).
Optimization of RNA extraction protocol for long-term archived formalin-fixed paraffin-embedded tissues of horses.
Exp Mol Pathol, 110, 104289.
https://doi.org/10.1016/j.yexmp.2019.104289 Publication
Researcher Affiliations
- Institute of Veterinary Pathology, Justus-Liebig-University, Gießen 35392, Germany. Electronic address: gisele.s.boos@vetmed.uni-giessen.de.
- Institute of Veterinary Pathology, Justus-Liebig-University, Gießen 35392, Germany.
- Unit for Biomathematics and Data Processing, Justus-Liebig-Universität, Gießen 35392, Germany.
- Institute of Virology, Philipps-University, Marburg 35043, Germany.
- Institute of Veterinary Pathology, Justus-Liebig-University, Gießen 35392, Germany; Center of Mind, Brain and Behavior, Justus-Liebig-University Gießen, Gießen, Germany.
MeSH Terms
- Animals
- Formaldehyde / chemistry
- Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / metabolism
- Horses
- Paraffin Embedding / methods
- Paraffin Embedding / veterinary
- RNA / analysis
- RNA / genetics
- RNA / isolation & purification
- Real-Time Polymerase Chain Reaction / methods
- Real-Time Polymerase Chain Reaction / veterinary
- Specimen Handling / standards
- Tissue Fixation / methods
- Tissue Fixation / veterinary
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