In vivo Plasmid Transfection and dsRNA Analysis

Subretinal injections (1 µL) were performed using a Pico-Injector (PLI-100, Harvard Apparatus). Plasmids were transfection in vivo using 10% Neuroporter (Genlantis). Immunolabeling was performed using antibodies against dsRNA (clone J2, English & Scientific Consulting), DICER1 (Santa Cruz Biotechnology), zonula occludens-1 (Invitrogen), Cre recombinase (EMD4Biosciences), or cleaved caspase-3 (Cell Signaling). dsRNA was isolated by immunoprecipitating homogenized tissue lysates with 40 µg of J2 for 16 h at 4 °C. Purified dsRNA was ligated to an anchor primer and purified by MinElute Gel extraction columns (Qiagen). Ligated dsRNA was denatured, reverse transcribed, and amplified by PCR. Amplified cDNA products were cloned into PCRII TOPO vector (Invitrogen) and sequenced. Homology to Alu consensus sequences was determined using CENSOR. Cell viability was assessed using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). Total RNA (1 µg) was reverse transcribed using qScript cDNA SuperMix (Quanta Biosciences) and amplified by real-time quantitative PCR (Applied Biosystems 7900 HT) with Power SYBR green Master Mix. Relative expressions were determined by the 2^−ΔΔCt method. miRNA abundance was quantified using All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia).
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.

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Kaneko H., Dridi S., Tarallo V., Gelfand B.D., Fowler B.J., Cho W.G., Kleinman M.E., Ponicsan S.L., Hauswirth W.W., Chiodo V.A., Karikó K., Yoo J.W., Lee D.K., Hadziahmetovic M., Song Y., Misra S., Chaudhuri G., Buaas F.W., Braun R.E., Hinton D.R., Zhang Q., Grossniklaus H.E., Provis J.M., Madigan M.C., Milam A.H., Justice N.L., Albuquerque R.J., Blandford A.D., Bogdanovich S., Hirano Y., Witta J., Fuchs E., Littman D.R., Ambati B.K., Rudin C.M., Chong M.M., Provost P., Kugel J.F., Goodrich J.A., Dunaief J.L., Baffi J.Z, & Ambati J. (2011). DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration. Nature, 471(7338), 325-330.

Publication 2011

Antibodies Assay Caspase 3 Cdna Cell proliferation Cell viability Cre recombinase Dicer1 Dsrna Homology sequences Mirna Plasmids Primer Quantitative real time pcr Sybr green Tissue Topo Transfection Vector Zonula occludens

Corresponding Organization : University of Kentucky

Other organizations : Yonsei University, University of Colorado Boulder, University of Florida, University of Pennsylvania, Sungkyunkwan University, Penn Presbyterian Medical Center, Meharry Medical College, Jackson Laboratory, Doheny Eye Institute, University of Southern California, Emory University, Australian National University, University of Sydney, UNSW Sydney, Howard Hughes Medical Institute, Rockefeller University, New York University, VA Salt Lake City Healthcare System, University of Utah, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Walter and Eliza Hall Institute of Medical Research, Centre hospitalier de l'Université Laval

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Variable analysis

independent variables

Subretinal injections (1 µL) using a Pico-Injector (PLI-100, Harvard Apparatus)
Plasmid transfection in vivo using 10% Neuroporter (Genlantis)

dependent variables

DsRNA isolated by immunoprecipitation and purified
Homology to Alu consensus sequences
Cell viability assessed using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega)
Relative gene expression determined by real-time quantitative PCR
MiRNA abundance quantified using All-in-One™ miRNA qRT-PCR Detection Kit (GeneCopoeia)

control variables

Immunolabeling performed using various antibodies

positive controls

DsRNA immunoprecipitation with 40 µg of J2 antibody for 16 h at 4 °C

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