Optimized methods for extracting circulating small RNAs from long-term stored equine samples.
Abstract: Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Furthermore, we showed that miRNA of NGS-sufficient quality can be recovered from blood samples after >10 years of storage at -80 °C. This allows retrospective analyses of miRNAs from archived samples.
Publication Date: 2016-06-29 PubMed ID: 27356979PubMed Central: PMC4928274DOI: 10.1186/s13028-016-0224-5Google Scholar: Lookup
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- Journal Article
Summary
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The research article focuses on the development and comparison of optimized methods for extracting small RNAs, specifically miRNAs, from stored equine serum and whole blood samples, offering a possible approach for future biomarker profiling in both human and veterinary medicine.
Objective of the Study
- The goal of this research is to systematically compare and optimize various methods for microRNA (miRNA) extraction from equine serum and whole blood samples. This is particularly significant as there is a lack of reliable and standardized methods for such extraction, especially from aged samples not treated with RNA stabilizing additives prior to freezing.
Methods of the Study
- The researchers compared the impact of short and long-term storage on various miRNA extraction methods, focusing on the effect of storage time at room temperature before freezing on serum samples.
- Non-stabilized samples were frozen and stored at -80 degrees Celsius for possible retrospective analyses, with some blood samples being stored for more than ten years.
Results and Conclusions
- The study found that storage time at room temperature prior to freezing did not affect the quality of miRNA in equine serum samples, indicating that miRNA is stable under these conditions.
- Furthermore, it was demonstrated that miRNA of Next Generation Sequencing (NGS) sufficient quality could be recovered from blood samples that had been stored at -80 degrees Celsius for over ten years.
- These results suggest the viability of performing retrospective analyses utilizing miRNA from archived samples, potentially contributing towards future routine biomarker profiling in various pathologic conditions.
Impact and Future Applications
- The findings of this research pave the way for the standardized extraction of miRNAs from long-term stored equine samples, providing a promising approach for future biomarker profiling in various pathological conditions in both human and veterinary medicine.
- The ability to extract miRNA of NGS-sufficient quality from samples stored for over ten years empowers scientists to conduct retrospective analyses, potentially offering deep insights into disease progression or effectiveness of long-term treatments.
Cite This Article
APA
Unger L, Fouché N, Leeb T, Gerber V, Pacholewska A.
(2016).
Optimized methods for extracting circulating small RNAs from long-term stored equine samples.
Acta Vet Scand, 58(1), 44.
https://doi.org/10.1186/s13028-016-0224-5 Publication
Researcher Affiliations
- Department of Clinical Veterinary Medicine, Swiss Institute of Equine Medicine, Vetsuisse Faculty, University of Bern and Agroscope, Länggassstrasse 124, 3012, Bern, Switzerland.
- Department of Clinical Veterinary Medicine, Swiss Institute of Equine Medicine, Vetsuisse Faculty, University of Bern and Agroscope, Länggassstrasse 124, 3012, Bern, Switzerland.
- Department of Clinical Research and Veterinary Public Health, Institute of Genetics, Vetsuisse Faculty, University of Bern, Bremgartenstrasse 109A, 3012, Bern, Switzerland.
- Department of Clinical Veterinary Medicine, Swiss Institute of Equine Medicine, Vetsuisse Faculty, University of Bern and Agroscope, Länggassstrasse 124, 3012, Bern, Switzerland.
- Department of Clinical Veterinary Medicine, Swiss Institute of Equine Medicine, Vetsuisse Faculty, University of Bern and Agroscope, Länggassstrasse 124, 3012, Bern, Switzerland. alicja.pacholewska@vetsuisse.unibe.ch.
- Department of Clinical Research and Veterinary Public Health, Institute of Genetics, Vetsuisse Faculty, University of Bern, Bremgartenstrasse 109A, 3012, Bern, Switzerland. alicja.pacholewska@vetsuisse.unibe.ch.
MeSH Terms
- Animals
- Blood Chemical Analysis / methods
- Freezing
- Horses
- MicroRNAs / blood
- MicroRNAs / isolation & purification
- Specimen Handling
References
This article includes 14 references
- Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function.. Cell 2004 Jan 23;116(2):281-97.
- Liang H, Gong F, Zhang S, Zhang CY, Zen K, Chen X. The origin, function, and diagnostic potential of extracellular microRNAs in human body fluids.. Wiley Interdiscip Rev RNA 2014 Mar-Apr;5(2):285-300.
- Buechli ME, Lamarre J, Koch TG. MicroRNA-140 expression during chondrogenic differentiation of equine cord blood-derived mesenchymal stromal cells.. Stem Cells Dev 2013 Apr 15;22(8):1288-96.
- van der Kolk JH, Pacholewska A, Gerber V. The role of microRNAs in equine medicine: a review.. Vet Q 2015 Jun;35(2):88-96.
- Desjardin C, Vaiman A, Mata X, Legendre R, Laubier J, Kennedy SP, Laloe D, Barrey E, Jacques C, Cribiu EP, Schibler L. Next-generation sequencing identifies equine cartilage and subchondral bone miRNAs and suggests their involvement in osteochondrosis physiopathology.. BMC Genomics 2014 Sep 17;15(1):798.
- Barrey E, Bonnamy B, Barrey EJ, Mata X, Chaffaux S, Guerin G. Muscular microRNA expressions in healthy and myopathic horses suffering from polysaccharide storage myopathy or recurrent exertional rhabdomyolysis.. Equine Vet J Suppl 2010 Nov;(38):303-10.
- Peiró-Chova L, Peña-Chilet M, López-Guerrero JA, García-Giménez JL, Alonso-Yuste E, Burgues O, Lluch A, Ferrer-Lozano J, Ribas G. High stability of microRNAs in tissue samples of compromised quality.. Virchows Arch 2013 Dec;463(6):765-74.
- Hammerle-Fickinger A, Riedmaier I, Becker C, Meyer HH, Pfaffl MW, Ulbrich SE. Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression analyses.. Biotechnol Lett 2010 Jan;32(1):35-44.
- Spornraft M, Kirchner B, Haase B, Benes V, Pfaffl MW, Riedmaier I. Optimization of extraction of circulating RNAs from plasma--enabling small RNA sequencing.. PLoS One 2014;9(9):e107259.
- Schwochow D, Serieys LE, Wayne RK, Thalmann O. Efficient recovery of whole blood RNA--a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species.. BMC Biotechnol 2012 Jun 27;12:33.
- Li X, Mauro M, Williams Z. Comparison of plasma extracellular RNA isolation kits reveals kit-dependent biases.. Biotechniques 2015 Jul;59(1):13-7.
- Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.. Anal Biochem 1987 Apr;162(1):156-9.
- Kirschner MB, Edelman JJ, Kao SC, Vallely MP, van Zandwijk N, Reid G. The Impact of Hemolysis on Cell-Free microRNA Biomarkers.. Front Genet 2013;4:94.
- Beekman JM, Reischl J, Henderson D, Bauer D, Ternes R, Peña C, Lathia C, Heubach JF. Recovery of microarray-quality RNA from frozen EDTA blood samples.. J Pharmacol Toxicol Methods 2009 Jan-Feb;59(1):44-9.
Citations
This article has been cited 9 times.- Hamza E, Cosandey J, Gerber V, Koch C, Unger L. The potential of three whole blood microRNAs to predict outcome and monitor treatment response in sarcoid-bearing equids.. Vet Res Commun 2023 Jan;47(1):87-98.
- Cosandey J, Hamza E, Gerber V, Ramseyer A, Leeb T, Jagannathan V, Blaszczyk K, Unger L. Diagnostic and prognostic potential of eight whole blood microRNAs for equine sarcoid disease.. PLoS One 2021;16(12):e0261076.
- Unger L, Abril C, Gerber V, Jagannathan V, Koch C, Hamza E. Diagnostic potential of three serum microRNAs as biomarkers for equine sarcoid disease in horses and donkeys.. J Vet Intern Med 2021 Jan;35(1):610-619.
- Unger L, Jagannathan V, Pacholewska A, Leeb T, Gerber V. Differences in miRNA differential expression in whole blood between horses with sarcoid regression and progression.. J Vet Intern Med 2019 Jan;33(1):241-250.
- Pacholewska A, Kraft MF, Gerber V, Jagannathan V. Differential Expression of Serum MicroRNAs Supports CD4⁺ T Cell Differentiation into Th2/Th17 Cells in Severe Equine Asthma.. Genes (Basel) 2017 Dec 12;8(12).
- Dini P, Loux SC, Scoggin KE, Esteller-Vico A, Squires EL, Troedsson MHT, Daels P, Ball BA. Identification of Reference Genes for Analysis of microRNA Expression Patterns in Equine Chorioallantoic Membrane and Serum.. Mol Biotechnol 2018 Jan;60(1):62-73.
- Loux SC, Scoggin KE, Bruemmer JE, Canisso IF, Troedsson MH, Squires EL, Ball BA. Evaluation of circulating miRNAs during late pregnancy in the mare.. PLoS One 2017;12(4):e0175045.
- Correia CN, Nalpas NC, McLoughlin KE, Browne JA, Gordon SV, MacHugh DE, Shaughnessy RG. Circulating microRNAs as Potential Biomarkers of Infectious Disease.. Front Immunol 2017;8:118.
- Pacholewska A, Mach N, Mata X, Vaiman A, Schibler L, Barrey E, Gerber V. Novel equine tissue miRNAs and breed-related miRNA expressed in serum.. BMC Genomics 2016 Oct 26;17(1):831.
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