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1 Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio, USA
* To whom correspondence should be addressed. E-mail: chandan.sen{at}osumc.edu.
For focal events like myocardial infarction, it is important to dissect infarction-induced biological responses as a function of space with respect to the infarct core. Laser microdissection pressure catapulting (LMPC) represents a recent variant of laser capture microdissection that enables robot-assisted rapid capture of catapulted tissue without direct user contact. This work represents the maiden effort to apply laser capture microdissection to study spatially-resolved biological responses in myocardial infarction. Infarcted areas of the surviving ischemia-reperfused murine heart was identified using a standardized hematoxylin QS staining procedure. Standard staining techniques fail to preserve tissue RNA. Exposure of the tissue to an aqueous media (typically used during standard immunohistochemical staining), with or without ribonuclease inhibitors, resulted in a rapid degradation of genes with ~80% loss in the first hour. Tissue elements (1 x 104 to 4 x 106 µm2) captured from the infarct and non-infarct sites with micron-level surgical precision were collected in a chaotropic RNA lysis solution. Isolated RNA was analyzed for quality employing microfluidics technology and reverse transcribed to generate high quality cDNA. Real-time PCR analysis of the cDNA showed marked (200- and 400-folds, respectively) induction of collagen Ia and IIIa at the infarct site compared to that in the non-infarct site. In sum, this work reports a sophisticated yet rapid approach to measure relative gene expressions from tissue elements captured from spatially resolved microscopic regions in the heart with micron-level precision.
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