Factors affecting RNA quantification from tissue long-term stored in formalin

Introduction: FFPE samples represent a rich pool of tissue samples for retrospective analyses of mRNA and miRNA analyses. However, the initial formalin fixation introduces a chemical modification of RNA and causes its degradation, therefore, a longer storage of tissue in formalin is predicted to render ribonucleic acids lost for isolation and subsequent analyses. Methods: Herein, we tested the impact of several factors on isolation of total RNA and detection with RT-qPCR from mouse liver tissue stored for over two years in formalin at room temperature. Results: The incubation of tissue in TAE buffer before RNA isolation yielded higher concentration and purity of RNA and also improved subsequent analyses. Using gene-specific primers for cDNA synthesis and shorter PCR products (under 70 bp) significantly improved RT-qPCR analyses. We also compared the level of expression and differential expression of mRNA and miRNA with matched fresh frozen liver tissue, and observed similar changes in differential expression of selected mRNA and miRNA as in matched fresh frozen tissue. Discussion: Conclusion is that adjustments of the protocol can substantially improve analyses of mRNA and miRNA from tissues stored in formalin for longer time.