Laser microdissection and pressure-catapulting technique to study gene expression in the reoxygenated myocardium.
For focal events such as 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 ischemic-reperfused murine heart were identified using a standardized hematoxylin QS staining procedure. Standard staining techniques fail to preserve tissue RNA. Exposure of the tissue to an aqueous medium (typically used during standard immunohistochemical staining), with or without RNase inhibitors, resulted in a rapid degradation of genes, with approximately 80% loss in the 1st h. Tissue elements (1 x 10(4)-4 x 10(6) microm(2)) captured from infarcted and noninfarcted sites with micrometer-level surgical precision were collected in a chaotropic RNA lysis solution. Isolated RNA was analyzed for quality by microfluidics technology and reverse transcribed to generate high-quality cDNA. Real-time PCR analysis of the cDNA showed marked (200- and 400-fold, respectively) induction of collagen Ia and IIIa at the infarcted site compared with the noninfarcted site. This work reports a sophisticated yet rapid approach to measurement of relative gene expressions from tissue elements captured from spatially resolved microscopic regions in the heart with micrometer-level precision.
Kuhn
Donald E
DE
Laboratory of Molecular Medicine, Department of Surgery, Davis Heart and Lung Research Institute, Ohio State University Medical Center, 473 W. 12th Avenue, Columbus, OH 43210, USA.
Roy
Sashwati
S
Radtke
Jared
J
Gupta
Sudip
S
Sen
Chandan K
CK
D000818:Animals; D003094:Collagen / Q000096:biosynthesis; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004210:Dissection; D015870:Gene Expression; D005821:Genetic Techniques / Q000295:instrumentation*; D006416:Hematoxylin; D007150:Immunohistochemistry; D007834:Lasers; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009203:Myocardial Infarction / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000378:metabolism*; D011312:Pressure; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012313:RNA; D003094:Collagen; D006416:Hematoxylin