Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyto ganglion cell and optic nerve development. Gradients of ephrinreceptors (Eph) create retinotopic maps that pattern RGC axonprojections to the brain, but little is known about the regulation ofthese gradients. In order to test the hypothesis that nasal/temporaldifferences in transcription factor expressions regulate EphA5 andEphA6 mRNA gradient pattern in RGCs, this initial study focuses onoptimizing laser capture microdissection (LCM) to yield high-qualityRNA for transcriptome sequencing.Methods: C57Bl/6 mice (postnatal day 2) eyes were enucleated andfrozen in molds with OCT compound with or without prior sucrosecryoprotection. Eyes were cryosectioned at 7µm and mounted ontoPEN-Membrane slides held at 4°C or room temperature. Sectionswere dehydrated prior to LCM of nasal and temporal thirds of theretinal ganglion cell layer (GCL). RNA quality was assessed usingRNA Nano and Pico chips (Agilent Bioanalyzer). Nasal/temporalsamples were tested in triplicate with quantitative reversetranscriptase PCR (qRT-PCR) and analyzed with Relative ExpressionSoftware Tool-MCSv2.Results: RNA quality was highest with flash frozen vs. sucrosecryoprotection of the eyes, with RNA Integrity Numbers (RINs) of7.6 and 5.9 respectively (scale 0 low-10 high). Mounting sections on4°C slides yielded better RNA quality than on room temperatureslides, with RINs of 8.8 and 7.8 respectively. Minimal RNAdegradation was detected within 90 mins following dehydration, withRINs declining from 8.8 to 8.5. qRT-PCR showed about a 3.5-foldenrichment of Pou4f2 mRNA in LCM samples vs. whole retina.There was about a 2-fold enrichment of EphA5 mRNA in temporalGCL vs. nasal GCL.Conclusions: LCM provides a powerful technique to extract highqualityRNA with sufficient yields for downstream, high-contentsequencing. Histological sections and RT-qPCR support anenrichment of RGC mRNA. Nasal/temporal differences in EphA5were detectable. Additional samples will be collected underoptimized conditions and analyzed prior to whole transcriptomeanalysis of the nasal and temporal GCL.Commercial Relationships: Steve Huynh, None; Deborah C.Otteson, NoneSupport: NIH R01 EY021792; NIH/NEI P30 EY007551Program Number: 2470 Poster Board Number: D0075Presentation Time: 2:45 PM - 4:30 PMA Non-Radioactive Assay for Measuring Retinal Base ExcisionRepair CapabilityVincent T. Ciavatta 1, 2 , Priscila P. Cunha 2 , Jeffrey H. Boatright 2 ,Sophia M. Tang 2 . 1 Rehabilitation R & D, Center of Excellence, USDept of Veterans Affairs, Decatur, GA; 2 Ophthalmology, EmoryUniversity School of Medicine, Atlanta, GA.Purpose: We are attempting to use endogenous retinal DNA repaircapability as part of a novel gene therapy approach. We aim toenhance DNA repair capability through physiological andpharmacological means. Oxoguanine glycosylase (OGG1) is anenzyme needed for repairing oxidized guanine residues, and it isexpressed in several mammalian retina cell types. To assess theimpact of our efforts, we developed a relatively inexpensive,fluorescence-based, non-radioactive assay to quantify OGG1 enzymeactivity.Methods: Methods - For substrate, a 50-base oligonucleotide with an8-oxodG residue at nucleotide (nt) position 26 and a fluorescentmoiety at the 5’ end was annealed to its complementary strand.Annealing (100 fmol/µL each oligonucleotide) was done according toLan et al., 2003. Protein extracts were prepared from fresh frozenC57BL6 mouse cortex and retina from C57BL6 and RD10 miceaccording to Bigot et al., 2009 and stored at -80°C. Cut reactionswere performed at 32°C for 1 h in 50 or 100 µL using 250 fmoldouble-stranded target, 5 to 100 µg protein extract, 50 mM HEPES(pH 7.6), 2 mM EDTA, 50 mM NaCl, 5% glycerol, and 0.1 mg/mLBSA. Reactions were stopped with 0.1 M NaOH and 37°C for 15min. DNA was recovered by ethanol precipitation, dissolved in 90%formamide, heat denatured, resolved by 7M urea, 15% PAGE, andDNA bands were photographed, digitized, and quantified.Results: Intensity of a 24 nt band showed a dose dependentrelationship with amount of retinal protein added up to 100 µg. Thediagnostic 24 nt band was detected in all retina and brain sampleswhen using the lowest amount of protein (5 µg). Omitting proteinproduced no detectable 24 nt band. Product was nearly eliminated if0.1 M NaOH was not used to stop the reaction. In weanling-agedmice, OGG1 activity was greater in C57BL6 than rd10 retina.Conclusions: This non-radioactive OGG1 assay is a uniquerefinement of one established OGG1 activity assay that usesradiolabeled substrates and another fluorescence-based method usinga hairpin, single-stranded oligonucleotide. Assay sensitivityapproximates that from the established methods. The assay isamenable to high throughput, fluorescence-based detection systemsand is useful for measuring effects of various independent variableson retinal OGG1 activity. Retinas undergoing degeneration may haveless DNA repair capability than wildtype retinas.Commercial Relationships: Vincent T. Ciavatta, None; Priscila P.Cunha, None; Jeffrey H. Boatright, None; Sophia M. Tang, NoneSupport: NEI R01 EY014026, NEI P30 EY06360, Research toPrevent Blindness (RPB), The Abraham & Phyllis Katz FoundationProgram Number: 2471 Poster Board Number: D0076Presentation Time: 2:45 PM - 4:30 PMEvolutionarily Conserved Minor Spliceosome is Required forDifferentiating Mouse Retinal NeuronsRahul N. Kanadia, Ashley M. Kilcollins. Physiology andNeurobiology, University of Connecticut, Storrs, CT.Purpose: Splicing removes introns and fuses exons, which isessential for eukaryotic gene expression and is carried out by themajor spliceosome. The major spliceosome consists of a core set ofsnRNAs including, U1, U2, U4, U5 and U6 that are required forsplicing. Interestingly, in eukaryotes, there exists anotherspliceosome called the minor spliceosome that is evolutionarilyconserved and consists of snRNAs including, U11, U12, U4atac andU6atac. Named thusly, for it removes introns in only 3% of thegenes. Given this small subset of introns that it regulates, we wantedto address the following questions. 1) Are the minor spliceosomeassociated snRNAs expressed in the developing retina? 2) Is theminor spliceosome function required for retinal development?Methods: We determined the expression of the minor spliceosomeassociated snRNAs by RT-PCR and in situ. We also employed P0 invivo retinal electroporation to inactivate U12 snRNA.Results: To test the presence of a functioning spliceosome in thedeveloping retina we examined the expression of U11 and U12snRNA. Surprisingly, U11 and U12 snRNAs were enriched in newlydifferentiating neurons, but absent in progenitor cells. Expression ofU11 and U12 was observed as distinct puncta in the nuclei of retinalganglion cells at P0, followed by amacrines and P4 and later in theONL at P10 and P14. Also, within the nuclei of the same cell, U11and U12 snRNAs do not overlap, which is surprising since they arethought to work as a di-snRNP. This might suggest that in the retinae,they function independently. Finally, inactivation of U12 snRNA didnot perturb the progenitor cell function, but it led to death ofterminally differentiating neurons. Specifically, the neuronal deathprogressed in the order in which they were differentiating. Forexample, amacrine cell death preceded the rod photoreceptor death.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. 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