Abstract
Laser-capture microdissection (LCM) coupled to downstream RNA analysis poses unique difficulties for the evaluation of mineralized tissues. A rapid protocol was thus developed to enable sufficient integrity of bone and cartilage tissue for reliable sectioning, while minimizing RNA loss associated with prolonged decalcification and purification steps. Specifically, the protocol involves pump-assisted, cardiac perfusion-fixation with paraformaldehyde, and moderate digestion of LCM-acquired tissue with proteinase K followed by DNase treatment and separation of RNA using magnetic beads. Reverse transcription and cDNA synthesis are performed immediately after RNA purification, without need for further protein removal.
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Acknowledgments
This work was supported by grants R0-1-MH061525 from the National Institutes of Health, RG-4503A4/1 from the National Multiple Sclerosis Society, and 2010-0913 from the Connecticut Department of Public Health to J.S.P, and grant R0-1-AR064381 from the National Institutes of Health to D.R. Xiaonan Xin and Xi Jiang contributed equally to this work.
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Xin, X., Jiang, X., Lichtler, A., Kronenberg, M., Rowe, D., Pachter, J.S. (2018). Laser-Capture Microdissection and RNA Extraction from Perfusion-Fixed Cartilage and Bone Tissue from Mice Implanted with Human iPSC-Derived MSCs in a Calvarial Defect Model. In: Murray, G. (eds) Laser Capture Microdissection. Methods in Molecular Biology, vol 1723. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7558-7_22
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DOI: https://doi.org/10.1007/978-1-4939-7558-7_22
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