dsrna mimetic pic  (InvivoGen)

Journal: bioRxiv

Article Title: Genome-Encoded Cytoplasmic Double-Stranded RNAs, Found in C9ORF72 ALS-FTD Brain, Provoke Propagated Neuronal Death

doi: 10.1101/248328

Figure Lengend Snippet: DsRNAmi localizes to the perinuclear region of the cytoplasm leading to neuronal death. Montage of Supplementary Movie 1. Every image contains the hour post transfection of GFP-positive neurons with Rhodamine-labeled dsRNAmi. (20x).

Article Snippet: Differentiated neurons were transfected with polyinosinic-polycytidylic acid (Poly(I:C)) HMW, a dsRNA mimetic (dsRNAmi) (catalog # tlrl-pic, Invivogen, San Diego, California) using Lipofectamine 2000 (catalog #11668027, Invitrogen, Waltham, MA) or Lipofectamine 2000 alone as a control, according to manufacturers protocol.

Techniques: Transfection, Labeling

Journal: bioRxiv

Article Title: Genome-Encoded Cytoplasmic Double-Stranded RNAs, Found in C9ORF72 ALS-FTD Brain, Provoke Propagated Neuronal Death

doi: 10.1101/248328

Figure Lengend Snippet: DsRNA is sufficient to induce neuronal cell death in differentiated human neurons and is present in the cerebelli of ALS/FTD patients with the C9ORF72 mutation. A) Western blots showing the induction of IFN-I signaling in differentiated human neurons (ReNcell VM) 24 hours post transfection with a dsRNAmi. B) Quantitation of neuronal viability using the CellTiter-Glo reagent to measure ATP levels 48 hours post transfection with a dsRNAmi at 0.25 (p=0.4807, n=4), 0.5 (p=0.891, n=4), 1 (p=0.0031, n=4), and 2 μg/mL (p=0.0005, n=4). Note that transfection of 2 μg/mL dsRNAmi causes significantly more neuronal death that 1 μg/mL (p=0.01, n=4). Immunohistochemistry with antibody specific to dsRNA (J2) on fixed thin sections of frontal cortex from: (C) normal control brains, (D) ALS patient with an SOD1 mutation, and (E) an ALS/FTD patient with a C9ORF72 mutation (E) (scale bar = 25 micron). Anti-dsRNA immunostaining on sections from cerebellum of (F) control and (G) ALS/FTD patient with C9ORF72 mutation. (scale bar = 10 micron). Insets digitally magnified 3 times. H) PCR amplification of cDNA with primers distal to the C9ORF72 expansion show a product made from RNA isolated by immunoprecipitation with an antibody specific for dsRNA from frontal cortex and cerebellum from ALS/FTD- patients with the C9ORF72 mutation, but not normal brains.

Article Snippet: Differentiated neurons were transfected with polyinosinic-polycytidylic acid (Poly(I:C)) HMW, a dsRNA mimetic (dsRNAmi) (catalog # tlrl-pic, Invivogen, San Diego, California) using Lipofectamine 2000 (catalog #11668027, Invitrogen, Waltham, MA) or Lipofectamine 2000 alone as a control, according to manufacturers protocol.

Techniques: Mutagenesis, Western Blot, Transfection, Quantitation Assay, Immunohistochemistry, Immunostaining, Amplification, Isolation, Immunoprecipitation

Journal: bioRxiv

Article Title: Genome-Encoded Cytoplasmic Double-Stranded RNAs, Found in C9ORF72 ALS-FTD Brain, Provoke Propagated Neuronal Death

doi: 10.1101/248328

Figure Lengend Snippet: DsRNA is sufficient to induce neuronal cell death in differentiated human cortical neurons. ReNcell CX (cortical neurons) were differentiated for two weeks and then transfected with 2 μ g/mL of dsRNAmi. The percent viability of cells was determined after two days using CellTiter-Glo reagent to measure ATP levels.

Article Snippet: Differentiated neurons were transfected with polyinosinic-polycytidylic acid (Poly(I:C)) HMW, a dsRNA mimetic (dsRNAmi) (catalog # tlrl-pic, Invivogen, San Diego, California) using Lipofectamine 2000 (catalog #11668027, Invitrogen, Waltham, MA) or Lipofectamine 2000 alone as a control, according to manufacturers protocol.

Techniques: Transfection

Journal: bioRxiv

Article Title: Genome-Encoded Cytoplasmic Double-Stranded RNAs, Found in C9ORF72 ALS-FTD Brain, Provoke Propagated Neuronal Death

doi: 10.1101/248328

Figure Lengend Snippet: Immunohistochemistry with antibody specific to dsRNA (J2) on fixed thin sections is specific to dsRNA. Anti-dsRNA antibody (J2) was added directly to tissue sections (A,C) or pre-incubated with dsRNAmi prior to application onto tissue sections (B,D). E) A membrane was blotted with either dsDNA or dsRNAmi at the same concentration. The anti-dsRNA antibody (J2) binds specifically to the dsRNAmi, but not the dsDNA.

Article Snippet: Differentiated neurons were transfected with polyinosinic-polycytidylic acid (Poly(I:C)) HMW, a dsRNA mimetic (dsRNAmi) (catalog # tlrl-pic, Invivogen, San Diego, California) using Lipofectamine 2000 (catalog #11668027, Invitrogen, Waltham, MA) or Lipofectamine 2000 alone as a control, according to manufacturers protocol.

Techniques: Immunohistochemistry, Incubation, Membrane, Concentration Assay