single-stranded (ss) dna  (Eurofins)

Journal: PLoS ONE

Article Title: Role of Interleukin 17 in Arthritis Chronicity through Survival of Synoviocytes via Regulation of Synoviolin Expression

doi: 10.1371/journal.pone.0013416

Figure Lengend Snippet: RA FLS were treated with SNP 0.01-1 µM for 24 h and apoptosis measured by SS DNA apoptosis kit ( A ). The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * P <0.05 compared to no treatment control. RA FLS were treated with SNP 0.01–1 µM for 24 h ( B, left panel ) or 0.1 µM over 24 h ( B, right panel ) and synoviolin expression measured by real-time RT-PCR. The results are expressed as the ratio of synoviolin/β-actin mRNA amplification, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * P <0.05 compared to no treatment control. RA FLS were pretreated with IL-17 100 ng/ml for 2 h then cotreated with SNP 0.1-1 µM for 24 h and apoptosis measured by SS DNA apoptosis kit ( C ). The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * P <0.05 compared to no treatment control. RA FLS were treated as above and annexin V analysed. Representative serial fluorescent microscopic pictures of Annexin V positive cells (green), double labelled with Propidium iodide (PI, red) at magnification x 200 ( D ).

Article Snippet: Apoptosis was also measured by a single stranded DNA (SS DNA) apoptosis ELISA kit (chemicon).

Techniques: Expressing, Quantitative RT-PCR, Amplification

Journal: PLoS ONE

Article Title: Role of Interleukin 17 in Arthritis Chronicity through Survival of Synoviocytes via Regulation of Synoviolin Expression

doi: 10.1371/journal.pone.0013416

Figure Lengend Snippet: To confirm specificity of siRNA knockdown of individual IL-17 receptors, RA FLS were nucleofected (amaxa) for 24 h with 0.5 µg IL-17RA (siRA), 0.05 µg IL-17RC (siRC) or siCONTROL siRNA (sictl) serum starved overnight then treated 50 ng/ml IL-17A for 8 h. IL-17RA or IL-17RC protein expression was measured by Western Blot. Membranes were stripped and reprobed for actin as a loading control ( A ). IL-17RA or IL-17RC mRNA expression was measured by real-time RT-PCR, normalised to GAPDH and expressed as percentage inhibition compared to sictl siRNA ( B ). * P <0.05, compared to sictl nucleofected RA FLS. RA FLS were nucleofected as above, serum starved overnight then pretreated with 50 ng/ml IL-17A for 2 h then cotreated with SNP 0.1 µM overnight and apoptosis measured by SS DNA apoptosis assays expressed as fold induction of apoptosis ( C ), n = 3 from 3 separate RA donors, mean ± SEM of duplicate experiments. * P <0.05, compared to sictl nucleofected RA FLS. RA FLS were nucleofected as above serum starved overnight then treated with 50 ng/ml IL-17A and ( D ) synoviolin levels analysed by real-time RT-PCR. Synoviolin mRNA level was normalized to GAPDH and expressed as fold increase over sictl siRNA treated samples, n = 5, mean ± SEM of duplicate experiments. * P <0.05, compared to sictl nucleofected RA FLS. † P <0.05 compared to IL-17 sictl treated samples. RA FLS were pretreated with 2 µg/ml of anti-IL-17RA or anti-IL-17RC Abs or isotype control goat Abs for 2 h then cotreated with IL-17 50 ng/ml for 24 h and synoviolin expression measured by real-time RT-PCR ( E ) , n = 3, mean ± SEM. * P <0.05, compared to untreated control. † P <0.05 compared to IL-17 treated samples.

Article Snippet: Apoptosis was also measured by a single stranded DNA (SS DNA) apoptosis ELISA kit (chemicon).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Inhibition

Journal: PLoS ONE

Article Title: Role of Interleukin 17 in Arthritis Chronicity through Survival of Synoviocytes via Regulation of Synoviolin Expression

doi: 10.1371/journal.pone.0013416

Figure Lengend Snippet: RA FLS were nucleofected (amaxa) for 24 h with 0.5 µg of 4 synoviolin siRNA duplexes (S-01 to S-04), synoviolin smartpool siRNA duplex or siCONTROL siRNA (sictl) and synoviolin expression analysed by real-time RT-PCR (A, left panel) or Western Blot (A, right panel). Synoviolin mRNA was normalized to GAPDH and results expressed as the percentage fold reduction compared to sictl treated samples, n = 5, mean ± SEM of duplicate experiments. * P <0.05, compared to sictl nucleofected RA FLS. ( B ), RA FLS were nucleofected as above then treated with SNP 0.1–1 µM overnight and apoptosis measured by the SS DNA apoptosis assay. Results are expressed as fold induction in apoptosis, n = 3 from 3 separate RA donors, mean ± SEM of duplicate experiments. * P <0.05, compared to sictl nucleofected RA FLS. ( C ), RA FLS were nucleofected as above, serum starved overnight then pretreated with 100 ng/ml IL-17A for 2 h then cotreated with SNP 0.1–1 µM overnight and apoptosis measured by SS DNA apoptosis assays expressed as fold induction of apoptosis, n = 3 from 3 separate RA donors, mean ± SEM of duplicate experiments. * P <0.05, compared to sictl nucleofected RA FLS. † P <0.05 compared to SNP treatment in S-08 nucleofected RA FLS.

Article Snippet: Apoptosis was also measured by a single stranded DNA (SS DNA) apoptosis ELISA kit (chemicon).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Apoptosis Assay

Journal: PLoS ONE

Article Title: Role of Interleukin 17 in Arthritis Chronicity through Survival of Synoviocytes via Regulation of Synoviolin Expression

doi: 10.1371/journal.pone.0013416

Figure Lengend Snippet: RA FLS were stimulated with IL-17A (50 or 100 ng/ml) and/or TNF (1 or 10 ng/ml) for 24 h and synoviolin mRNA expression measured by real-time RT-PCR ( A ). The results based on a ratio of synoviolin/β-actin mRNA amplification are presented as the fold induction in synoviolin mRNA expression relative to control samples, n = 3, mean ± SEM. * P <0.05 compared to no treatment control. † P <0.05 compared to TNF treated samples. RA FLS were pretreated with IL-17 100 ng/ml and TNF 10 ng/ml for 2 h then cotreated with SNP 0.5 µM ( B, left panel ) or 0.1 µM ( B, right panel ) for 24 h and apoptosis measured by SS DNA apoptosis kit. The results are presented as the fold induction in apoptosis relative to control samples, n = 3, mean ± SEM of duplicate experiments from 3 different RA donors. * P <0.05 compared to SNP treatment in S-08 nucleofected RA FLS.

Article Snippet: Apoptosis was also measured by a single stranded DNA (SS DNA) apoptosis ELISA kit (chemicon).

Techniques: Expressing, Quantitative RT-PCR, Amplification

Journal: Journal of Analytical Methods in Chemistry

Article Title: Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

doi: 10.1155/2016/4681421

Figure Lengend Snippet: Biotinylation of deoxyguanosine at an abasic site in double-stranded DNA oligonucleotides. The biotinylation of the unpaired deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal ( 1 ).

Article Snippet: The 100-mer single-stranded DNA oligonucleotides (SS-G), 5′-(CAG TGA AGT TGG CAG ACT GAG CCA GGT CCC ACA GAT GCA GTG ACC GGA GTC ATT GCC AAA CTC TGC AGG AGA GCA AGG GCT GTC TAT AGG TGG CAA GTC A)-3′, were custom-synthesized (GeneDesign, Japan).

Techniques: Conjugation Assay

Journal: Annals of Transplantation

Article Title: Pretreatment with a Phosphodiesterase-3 Inhibitor, Milrinone, Reduces Hepatic Ischemia-Reperfusion Injury, Minimizing Pericentral Zone-Based Liver and Small Intestinal Injury in Rats

doi: 10.12659/AOT.922306

Figure Lengend Snippet: Assessment of single-stranded deoxyribonucleic acid (ss-DNA) expression in the small intestine: The sham group ilea contained no strongly stained ss-DNA-positive cells. The ischemia-reperfusion (IR) group showed some cells that were strongly positive for ss-DNA labeling (black arrows), mainly at the lamina propria, with congestion. Localization of the ss-DNA expression was similar to that of the cleaved caspase-3 expression ( A ). The IR+milrinone group had fewer of these cells in the small intestines than did the IR group, but the difference was not significant ( B ).

Article Snippet: Anti-single-stranded deoxyribonucleic acid (ss-DNA) antibody (1: 50; Immuno-Biological Laboratories, Fujioka, Gunma, Japan) was used as a marker of apoptotic DNA fragmentation.

Techniques: Expressing, Staining, DNA Labeling