Sprouting transcriptome in cortical neurons: young

Selective isolation of total RNA and then whole genome expression analysis of sprouting neurons in peri-infarct cortex of the adult rat after stroke, compared to adjacent neurons that have not sprouted a new connection, in young adult (2 months) and aged (2 years) animals. Two different fluorescent conjugates of the tracer cholera toxin B (CTb), CTb-Alexa 488 and CTb-Alexa 647 (Molecular Probes), were sequentially injected into peri-infarct cortex at the sites of post-stroke axonal sprouting using a picospritzer pressure injection system. After survival periods of 7 days and 21 days (separate cohorts of animals), CTb-647 was injected exactly within CTb-488 injection site. Seven days after the second tracer injection, laser capture microdissection (LCM) was used to capture ~300 neurons for each cell type. Total RNA was isolated from captured cells and subjected to two rounds of T7 amplification.