E-MEXP-1387 - Transcription profiling by array of rat retina after intra-orbital nerve transection, intra-orbital nerve crush or no treatment
Released on 14 June 2008, last updated on 22 August 2014
Array experiment: Animals were divided into a control group (naive), which did not undergo any experimental manipulation (n=20) and two experimental groups, one receiving an intra-orbital nerve transection (IONT, n=60) and the other an intra-orbital nerve crush (IONC, n=60) Sterile precautions were maintained for all surgical procedures. The left ON was intraorbitally axotomized according to procedures that are standard in our laboratory. (REF) Briefly, an incision was made in the superior orbital rim, the superoexternal orbital contents were dissected, and the superior and external rectus muscles were removed. When performing IONT, the dura mater was opened longitudinally to spare the blood supply, and the optic nerve was transected 0.5 mm from the optic disc. To perform IONC the optic nerve was crushed 3 mm from the optic disc during 10 seconds using a pair of watchmakerÌÄåâÌâåÕs forceps (Vidal-Sanz et al., 1988, 1991; Villegas-PÌÄåÄÌâå©rez et al., 1993). Before and after the procedure, the eye fundus was observed through the operating microscope to assess the integrity of the retinal blood flow. Tissue processing and RNA extraction: Animals were kept the appropriate time post injury (12h, 24h, 48h, 3d 7d and 15d, n=8-12 per time point) and then sacrificed by an intraperitoneal overdose of sodium pentobarbital. Left retinas were fresh dissected and immediately frozen in liquid nitrogen. Four retinas from each time point, animal group and biological replica were pooled and RNA was extracted using Trizol (Invitrogen). RNA was further cleaned through RNAeasy mini kit columns (Quiagen). The RNA integrity was checked using a Bioanalyzer (Agilent Technologies, USA) and concentrations were determined using a NanoDrop (NanoDrop Technologies, Wilmington, DE, USA).
transcription profiling by array, injury design, time series design
Time course profiling of the retinal transcriptome after optic nerve transection and optic nerve crush. Agudo M, PŽrez-Mar’n MC, Lšnngren U, Sobrado P, Conesa A, C‡novas I, Salinas-Navarro M, Miralles-Imperial J, Hallbššk F, Vidal-Sanz M.