antisense dna oligo probes  (LGC Biosearch)

Journal: Arthritis and Rheumatism

Article Title: A secretagogue-siRNA conjugate confers resistance to cytotoxicity in a cell model of Sj?gren's syndrome

doi: 10.1002/art.30450

Figure Lengend Snippet: Carbachol and siRNA portions of conjugate retained function after conjugation. (A) Scheme presenting the two-step activation and conjugation of choline to 5’-amino-modified RNA. Activation of choline was carried out by reaction with 4-nitrophenyl chloroformate followed by reaction with amino-modified RNA. (B) Caspase-3 gene expression after transfection of siRNA, conjugate, or negative control conjugate. Free siCaspase-3 treatment indicates naked siRNA targeting caspase-3 was added to the cells in the absence of transfecting reagent. Negative control conjugate contains a scrambled siRNA sequence. Asterisks indicate p<0.05 compared to negative control conjugate as determined by one-way ANOVA with Dunnett's multiple comparison test. (C) Conjugate was added to HSG cells as indicated and intracellular calcium release was measured as fluorescence intensity. Bars represent mean with standard error (n=3 independent experiments), and dashed line indicates level of background fluorescence. (D) Calcium imaging of Parc5 cells treated with 100μM carbachol, a free siRNA control, or 8.7μM conjugate. Each colored line represents calcium response in a single cell.

Article Snippet: The FAM-labeled DNA oligo probe specific for the antisense strand of caspase-3 siRNA (Exiqon Woburn, MA) was diluted to 80nM in PBS and heated to 80°C for two minutes before addition to the cells.

Techniques: Conjugation Assay, Activation Assay, Modification, Expressing, Transfection, Negative Control, Sequencing, Fluorescence, Imaging

Journal: Arthritis and Rheumatism

Article Title: A secretagogue-siRNA conjugate confers resistance to cytotoxicity in a cell model of Sj?gren's syndrome

doi: 10.1002/art.30450

Figure Lengend Snippet: Conjugate detected in HSG cell cytoplasm within 30 minutes of incubation. HSG cells were treated with 5μM conjugate for the indicated times before being fixed and used for in situ hybridization with a FAM-labeled DNA oligo probe specific for the antisense strand of caspase-3 siRNA. Arrows indicate conjugate detected in cytoplasm of cells (green). Nuclei counterstained with DAPI (blue). Images shown at 200X magnification.

Article Snippet: The FAM-labeled DNA oligo probe specific for the antisense strand of caspase-3 siRNA (Exiqon Woburn, MA) was diluted to 80nM in PBS and heated to 80°C for two minutes before addition to the cells.

Techniques: Incubation, In Situ Hybridization, Labeling

Journal: Arthritis and Rheumatism

Article Title: A secretagogue-siRNA conjugate confers resistance to cytotoxicity in a cell model of Sj?gren's syndrome

doi: 10.1002/art.30450

Figure Lengend Snippet: Conjugate treatment reduced caspase-3 mRNA expression in HSG cells. A) Conjugate had comparable caspase-3 knockdown efficiency compared to transfected siRNA. HSG cells were treated with 8.71μM conjugate or negative control conjugate and incubated for 48 hours. Caspase-3 gene expression was then analyzed by qRT-PCR. Asterisks (*) indicate p<0.01 compared to negative control conjugate treated cells as determined by one-way ANOVA with Dunnett's multiple comparison test. Bars represent mean with standard error (n=5 independent experiments). B) Conjugate had no effect on M3R-negative cells. HeLa cells were treated with conjugate or transfected with caspase-3 siRNA, incubated for 72 hours, and caspase-3 gene expression was analyzed by qRT-PCR. Bars represent mean with standard error (n=2). C) Conjugate incubation did not induce interferon response.HSG cells were treated with conjugate or transfected with caspase-3 siRNA as previously described and incubated for 72 hours. qRT-PCR was used to analyze mRNA expression of interferon response genes OAS1 (white bars) and MX1 (black bars) which remained unchanged. Bars represent mean with standard error (n=3).

Article Snippet: The FAM-labeled DNA oligo probe specific for the antisense strand of caspase-3 siRNA (Exiqon Woburn, MA) was diluted to 80nM in PBS and heated to 80°C for two minutes before addition to the cells.

Techniques: Expressing, Transfection, Negative Control, Incubation, Quantitative RT-PCR

Journal: Arthritis and Rheumatism

Article Title: A secretagogue-siRNA conjugate confers resistance to cytotoxicity in a cell model of Sj?gren's syndrome

doi: 10.1002/art.30450

Figure Lengend Snippet: Conjugate treatment reduced caspase-3 protein expression in HSG cells. HSG cells were treated with 8.71μM conjugate and incubated for 72 hours. Caspase-3 protein expression was analyzed by immunofluorescence (A) using rabbit anti-caspase-3 antibodies and alexa fluor 568 goat anti-rabbit IgG secondary antibodies (red). Cell nuclei were counterstained with DAPI (blue), and caspase-3 siRNA or conjugate was detected by in situ hybridization (green). Images shown at 100X magnification. (B) Quantitation of caspase-3 protein levels were measured using Image J image analysis software and normalized to untreated cells. Asterisks (*) indicate p<0.05 as determined by student t test. Bars represent mean with standard error (n=3 independent experiments). (C) Lysates of cells treated as described above were analyzed for caspase-3 and GAPDH protein expression by Western blot.

Article Snippet: The FAM-labeled DNA oligo probe specific for the antisense strand of caspase-3 siRNA (Exiqon Woburn, MA) was diluted to 80nM in PBS and heated to 80°C for two minutes before addition to the cells.

Techniques: Expressing, Incubation, Immunofluorescence, In Situ Hybridization, Quantitation Assay, Software, Western Blot

Journal: Arthritis and Rheumatism

Article Title: A secretagogue-siRNA conjugate confers resistance to cytotoxicity in a cell model of Sj?gren's syndrome

doi: 10.1002/art.30450

Figure Lengend Snippet: Conjugate treatment prevented TNF-α induced apoptosis in HSG cells. HSG cells were treated with 10 μM conjugate, 10μM negative control conjugate, or transfected with caspase-3 siRNA and incubated for 96 hours. The cells were then treated with TNF-α (50ng/ml) and cycloheximide (10μg/ml) for eight hours and stained with Annexin-V and propidium iodide. (A) Flow cytometry was used to assess early apoptotic cells (Annexin-V positive) and (B) late apoptotic cells (Annexin-V/PI positive) in treated (black bars) and untreated (white bars) cells. The percent of early and late apoptotic cells after TNF-α/cycloheximide treatment was significantly reduced in caspase-3-transfected and conjugate-treated cells compared to cells treated with negative control conjugate (asterisks p<0.01 as determined by t test). Bars represent mean with standard error (n=4 independent experiments).

Article Snippet: The FAM-labeled DNA oligo probe specific for the antisense strand of caspase-3 siRNA (Exiqon Woburn, MA) was diluted to 80nM in PBS and heated to 80°C for two minutes before addition to the cells.

Techniques: Negative Control, Transfection, Incubation, Staining, Flow Cytometry