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Updated: Jul 10, 2026
Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species
Published on: May 19, 2018
RNA isolation and quantitative PCR from HOPE- and formalin-fixed bovine lymph node tissues
Jaydene Witchell1, Dhaval Varshney, Trusha Gajjar
1School of Life Sciences, University of Hertfordshire, Hatfield, Herts AL10 9AB, UK.
Hepes glutamic acid buffer-mediated organic solvent protection effect (HOPE) fixation yields superior ribonucleic acid (RNA) for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) from cattle lymph nodes. HOPE-fixed tissues provide higher RNA integrity and quantity compared to formalin fixation, enabling more reliable gene expression analysis.
Area of Science:
- Molecular Biology
- Veterinary Pathology
- Biochemistry
Background:
- Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is crucial for gene expression analysis.
- Formalin fixation is a common method for preserving tissues but can degrade RNA.
- Hepes glutamic acid buffer-mediated organic solvent protection effect (HOPE) fixation is a newer method for tissue preservation.
Purpose of the Study:
- To compare the efficacy of HOPE fixation versus formalin fixation for RNA extraction and subsequent qRT-PCR analysis.
- To evaluate RNA yield and integrity from both fixation methods in Mycobacterium bovis-infected cattle lymph nodes.
- To determine the suitability of HOPE-fixed tissues for reliable gene expression studies.
Main Methods:
- Total RNA was extracted from both formalin-fixed and HOPE-fixed, paraffin-embedded cattle lymph node tissues using a commercial kit and the Trizol method.
- RNA quantity and integrity were assessed via spectrophotometry and electrophoresis.
- qRT-PCR was performed to quantify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels.
Main Results:
- RNA extracted from HOPE-fixed tissues showed high quantity and integrity, regardless of the extraction method (commercial kit or Trizol).
- RNA extracted from formalin-fixed tissues using the commercial kit had comparable quantity but compromised integrity.
- RNA extraction from formalin-fixed tissues using Trizol was unsuccessful, and qRT-PCR yielded significantly lower target mRNA levels from formalin-fixed tissues compared to HOPE-fixed tissues.
Conclusions:
- HOPE fixation is a superior method for preserving RNA in cattle lymph node tissues for qRT-PCR analysis compared to formalin fixation.
- The improved RNA integrity and quantity from HOPE-fixed tissues lead to more accurate gene expression quantification.
- HOPE fixation offers a viable alternative for molecular studies requiring high-quality RNA from fixed tissues.
