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. 2024 Apr;76(4):153-160.
doi: 10.2144/btn-2023-0092. Epub 2024 Feb 9.

Extracting high-quality RNA from formaldehyde-fixed naturally aged neuromusculoskeletal tissues

Seth D Thompson  1   2   3 , Rajeswari Pichika  1   2   4 , Richard L Lieber  1   2   4 , Mitra Lavasani  1   2   3
Affiliations

Extracting high-quality RNA from formaldehyde-fixed naturally aged neuromusculoskeletal tissues

Seth D Thompson et al. Biotechniques. 2024 Apr.

Abstract

Modern approaches to discovering molecular mechanisms and validating treatments for age-related neuromusculoskeletal dysfunction typically rely on high-throughput transcriptome analysis. Previously harvested and fixed tissues offer an incredible reservoir of untapped molecular information. However, obtaining RNA from such formaldehyde-fixed neuromusculoskeletal tissues, especially fibrotic aged tissues, is technically challenging and often results in RNA degradation, chemical modification and yield reduction, prohibiting further analysis. Therefore, we developed a protocol to extract high-quality RNA from formaldehyde-fixed brain, cartilage, muscle and peripheral nerve isolated from naturally aged mice. Isolated RNA produced reliable gene expression data comparable to fresh and flash-frozen tissues and was sensitive enough to detect age-related changes, making our protocol valuable to researchers in the field of aging.

Keywords: RNA isolation from fixed tissue; aging; formaldehyde fixation; gene expression; neuromusculoskeletal tissues; tissue bank.

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Conflict of interest statement

The authors have no competing interests or relevant affiliations with any organization or entity with an interest in or conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Figures

Figure 1.
Figure 1.. Assessment of amplified mRNA by quantitative polymerase chain reaction.
Amplification curves of the housekeeping gene Gapdh obtained from naturally aged (24-month-old) mouse brain, articular cartilage, gastrocnemius muscle and sciatic nerve using fresh (purple), flash-frozen (orange) and formaldehyde-fixed (green) preservation methods. Dark green horizontal lines on each graph indicate threshold level. RFU: Relative fluorescence unit.
Figure 2.
Figure 2.. Postamplification melting curves.
Graphs showing the melting peaks of the housekeeping gene Gapdh from fresh (purple), flash-frozen (orange) and formaldehyde-fixed (green) preservation methods in naturally aged (24-month-old) mouse brain, sciatic nerve, articular cartilage and gastrocnemius tissues. Dark green horizontal lines on each graph indicate threshold level. -d(RFU)/dt: The negative first derivative of change in fluorescence as a function of temperature; RFU: Relative fluorescence unit.
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References

    1. Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nat. Protoc. 1(2), 581–585 (2006). - PubMed
    1. Foss RD, Guha-Thakurta N, Conran RM, Gutman P. Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls. Diagn. Mol. Pathol. 3(3), 148–155 (1994). - PubMed
    1. Hoffman EA, Frey BL, Smith LM, Auble DT. Formaldehyde crosslinking: a tool for the study of chromatin complexes. J. Biol. Chem. 290(44), 26404–26411 (2015). - PMC - PubMed
    1. Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K. Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res. 27(22), 4436–4443 (1999). - PMC - PubMed

    2. •• First study to demonstrate the specific modifications that occur to RNA in tissues fixed with formaldehyde.

    1. Soles A, Selimovic A, Sbrocco K et al. Extracellular matrix regulation in physiology and in brain disease. Int. J. Mol. Sci. 24(8), 7049 (2023). - PMC - PubMed

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