Xin Xiaonan, Jiang Xi, Lichtler Alexander, Kronenberg Mark, Rowe David, Pachter Joel S
Center for Regenerative Medicine and Skeletal Development, School of Dental Medicine, UConn Health, Farmington, CT, USA.
Department of Reconstructive Sciences, School of Dental Medicine, UConn Health, Farmington, CT, USA.
Methods Mol Biol. 2018;1723:385-396. doi: 10.1007/978-1-4939-7558-7_22.
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.
激光捕获显微切割（LCM）与下游RNA分析相结合，在评估矿化组织时带来了独特的困难。因此，开发了一种快速方案，以确保骨和软骨组织具有足够的完整性，以便进行可靠的切片，同时将与长时间脱钙和纯化步骤相关的RNA损失降至最低。具体而言，该方案包括用多聚甲醛进行泵辅助心脏灌注固定，用蛋白酶K对LCM获取的组织进行适度消化，然后进行DNase处理，并使用磁珠分离RNA。RNA纯化后立即进行逆转录和cDNA合成，无需进一步去除蛋白质。
