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
| Autor: | Alexander C. Lichtler, Xi Jiang, David W. Rowe, Mark S. Kronenberg, Joel S. Pachter, Xiaonan Xin |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Induced Pluripotent Stem Cells Laser Capture Microdissection Bone tissue Article Bone and Bones Mice 03 medical and health sciences 0302 clinical medicine medicine Animals Humans Microdissection Laser capture microdissection Bone decalcification Chemistry Gene Expression Profiling Cartilage Skull RNA Mesenchymal Stem Cells Reverse transcriptase Cell biology Perfusion Disease Models Animal 030104 developmental biology medicine.anatomical_structure 030220 oncology & carcinogenesis RNA extraction |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493975570 |
| DOI: | 10.1007/978-1-4939-7558-7_22 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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