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.

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.