Quantitative real-time PCR for equine cytokine mRNA in nondecalcified bone tissue embedded in methyl methacrylate.
Abstract: Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and embedded in a hydrophobic acrylic resin. Total RNA was extracted from defined regions of single 50 microm sawed sections. These RNA preparations are suitable for quantitative PCR analysis of mRNA of different cytokines. In addition, the universally expressed housekeeping GAPDH mRNA proved to be useful as an amplification control and to correct for the degree of RNA degradation, which may vary considerably among samples. Reverse transcribed mRNA was amplified and quantitated in Real-Time PCR using a fluorescein labeled internal TaqMan probe.
Publication Date: 1999-12-14 PubMed ID: 10541764DOI: 10.1007/s002239900717Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study presents a method for quantifying specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and embedded in a hydrophobic acrylic resin, enabling the quantitative PCR analysis of cytokine mRNA in equines.
Research Overview
- The research focuses on a method for extracting and quantifying nucleic acid sequences from bone tissues and calcified tissues embedded in hydrophobic acrylic resin. Previously, this kind of analysis had been successful on DNA and RNA sequences extracted from tissues embedded in paraffin only.
- The method introduced in the research involves the extraction of total RNA from defined regions of single sections of an undecalcified bone, which were fifty micrometers apart. Paraformaldehyde was used as the fixative, and a hydrophobic acrylic resin was used as the embedding medium.
Utilization of Total RNA and Real-Time PCR in the Research
- The total RNA preparations extracted from the bone sections were found suitable for quantitative PCR analysis of different cytokine mRNAs.
- This new method of mRNA sequence identification thus provides a potential for studying gene expression at the molecular level, especially for those genes expressed in hard, calcified tissues.
<h3 role of GAPDH mRNA
- The research identifies Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, which is universally expressed, as an effective amplification control. This element also helps in correcting the degree of RNA degradation which may vary across different samples.
- Its presence in all RNA samples makes it a useful reference to normalize the expression levels of the target mRNAs of interest.
Use of Fluorescein Labeled Internal TaqMan Probe
- In this study, the reverse-transcribed mRNA was amplified and quantified using a Real-Time PCR process with a fluorescein labeled internal TaqMan probe.
- The use of the fluorescein-labeled probe highlights the specific mRNA sequences in real time, thus enabling easier and more accurate mRNA quantitation.
Cite This Article
APA
Leutenegger CM, von Rechenberg B, Huder JB, Zlinsky K, Mislin C, Akens MK, Auer J, Lutz H.
(1999).
Quantitative real-time PCR for equine cytokine mRNA in nondecalcified bone tissue embedded in methyl methacrylate.
Calcif Tissue Int, 65(5), 378-383.
https://doi.org/10.1007/s002239900717 Publication
Researcher Affiliations
- Musculosceletal Research Unit, Department of Veterinary Surgery, University of Zurich, Switzerland.
MeSH Terms
- Animals
- Bone and Bones / chemistry
- Cytokines / analysis
- Cytokines / genetics
- DNA / analysis
- Electrophoresis, Agar Gel
- Formaldehyde
- Glyceraldehyde-3-Phosphate Dehydrogenases / analysis
- Horses
- Methacrylates
- Nitric Oxide Synthase / analysis
- Polymerase Chain Reaction / methods
- Polymers
- RNA, Messenger / analysis
- Tissue Embedding
- Tissue Fixation
Citations
This article has been cited 13 times.- Bouaicha S, von Rechenberg B, Osterhoff G, Wanner GA, Simmen HP, Werner CM. Histological remodelling of demineralised bone matrix allograft in posterolateral fusion of the spine--an ex vivo study.. BMC Surg 2013 Dec 13;13:58.
- Starke A, Corsenca A, Kohler T, Knubben J, Kraenzlin M, Uebelhart D, Wüthrich RP, von Rechenberg B, Müller R, Ambühl PM. Correction of metabolic acidosis with potassium citrate in renal transplant patients and its effect on bone quality.. Clin J Am Soc Nephrol 2012 Sep;7(9):1461-72.
- Heidenreich D, Langhoff JD, Nuss K, Kluge K, Kämpf K, Zlinsky K, Hilbe M, Mayer J, von Rechenberg B. The use of BoneWelding® technology in spinal surgery: an experimental study in sheep.. Eur Spine J 2011 Nov;20(11):1821-36.
- Espino AM, Rivera F. Quantitation of cytokine mRNA by real-time RT-PCR during a vaccination trial in a rabbit model of fascioliasis.. Vet Parasitol 2010 Apr 19;169(1-2):82-92.
- Kuttenberger JJ, Waibel A, Stübinger S, Werner M, Klasing M, Ivanenko M, Hering P, von Rechenberg B, Sader R, Zeilhofer HF. Bone healing of the sheep tibia shaft after carbon dioxide laser osteotomy: histological results.. Lasers Med Sci 2010 Mar;25(2):239-49.
- Allen CA, Payne SL, Harville M, Cohen N, Russell KE. Validation of quantitative polymerase chain reaction assays for measuring cytokine expression in equine macrophages.. J Immunol Methods 2007 Dec 1;328(1-2):59-69.
- Berger J, Valdez S, Puschner B, Leutenegger CM, Gardner IA, Madigan JE. Effects of oral tetrachlorvinphos fly control (Equitrol) administration in horses: physiological and behavioural findings.. Vet Res Commun 2008 Jan;32(1):75-92.
- Bogaert L, Van Poucke M, De Baere C, Peelman L, Gasthuys F, Martens A. Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids.. BMC Biotechnol 2006 Apr 27;6:24.
- Waselau AC, Nadler D, Müller JM, Zlinszky K, Hilbe M, Auer JA, von Rechenberg B. The effect of cartilage and bone density of mushroom-shaped, photooxidized, osteochondral transplants: an experimental study on graft performance in sheep using transplants originating from different species.. BMC Musculoskelet Disord 2005 Dec 15;6:60.
- Jin S, Chen C, Montelaro RC. Equine infectious anemia virus Gag p9 function in early steps of virus infection and provirus production.. J Virol 2005 Jul;79(14):8793-801.
- Dawson HD, Beshah E, Nishi S, Solano-Aguilar G, Morimoto M, Zhao A, Madden KB, Ledbetter TK, Dubey JP, Shea-Donohue T, Lunney JK, Urban JF Jr. Localized multigene expression patterns support an evolving Th1/Th2-like paradigm in response to infections with Toxoplasma gondii and Ascaris suum.. Infect Immun 2005 Feb;73(2):1116-28.
- Tadesse Y, Bernier L, Hintz WE, Horgen PA. Real time RT-PCR quantification and Northern analysis of cerato-ulmin ( CU) gene transcription in different strains of the phytopathogens Ophiostoma ulmi and O. novo-ulmi.. Mol Genet Genomics 2003 Sep;269(6):789-96.
- Leutenegger CM, Mislin CN, Sigrist B, Ehrengruber MU, Hofmann-Lehmann R, Lutz H. Quantitative real-time PCR for the measurement of feline cytokine mRNA.. Vet Immunol Immunopathol 1999 Nov 30;71(3-4):291-305.
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