cdna cloning primer  (Integrated DNA Technologies)

Journal: PLoS ONE

Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome

doi: 10.1371/journal.pone.0030414

Figure Lengend Snippet: Characteristics and gene expressions before and after weight loss in obese patients (1 st cohort).

Article Snippet: Furthermore, cDNA clones (OriGene) for IRAK3 (TNFAIP3 and SOD2) were used to double check the primer specificity.

Techniques:

Journal: PLoS ONE

Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome

doi: 10.1371/journal.pone.0030414

Figure Lengend Snippet: ( A ) A structural model containing TLR2 as cell surface marker, NFκB as transcription factor, TNFα as inflammatory output, SOD2 as oxidative stress marker, and IRAK3 and TNFAIP3 as putative inhibitors was determined by promoter and gene annotation analysis of deregulated genes. Flow of the pathway at the protein interaction level is indicated by black arrows. Blunted arrows indicate inhibition. Phosphorylation is indicated by ???. Note that NFκB is constitutively bound to IκB molecules, which confine its localization to the cytosol. IKK complex phosphorylation of IκB promotes its degradation, thereby freeing NFκB to enter the nucleus and activate transcription of target genes. Gene expression of key molecules in the TLR2/NFκB inflammatory pathway was measured by qRT-PCR in blood monocytes of ( B ) the first cohort comprising 14 lean controls and 21 obese patients, and ( C ) the second cohort comprising 25 lean controls and 102 obese patients. Data are expressed as means. ** P <0.01 and *** P <0.001 obese persons compared with lean controls.

Article Snippet: Furthermore, cDNA clones (OriGene) for IRAK3 (TNFAIP3 and SOD2) were used to double check the primer specificity.

Techniques: Marker, Inhibition, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome

doi: 10.1371/journal.pone.0030414

Figure Lengend Snippet: Association of RNA expressions in monocytes and blood levels with occurrence of metabolic syndrome.

Article Snippet: Furthermore, cDNA clones (OriGene) for IRAK3 (TNFAIP3 and SOD2) were used to double check the primer specificity.

Techniques:

Journal: PLoS ONE

Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome

doi: 10.1371/journal.pone.0030414

Figure Lengend Snippet: ( A ) Gene expression was analyzed by measuring relative RNA levels using qRT-PCR, protein expression and ROS production were determined by flow cytometry in THP-1 cells exposed to 1 or 10 µg/ml gADIPOQ (n = 6) or in IRAK3 -depleted THP-1 cells exposed to 10 µg/ml gADIPOQ (n = 4) for 6 h and 24 h. Data shown are means ± SEM of 24 h exposed cells normalized to 6 h exposed cells. * P <0.05, ** P <0.01 and *** P <0.001 compared with THP-1 cells exposed to high gADIPOQ; $$ P <0.01 and $$$ P <0.001 compared with THP-1 cells exposed to low gADIPOQ. ( B ) Gene/protein expression in THP-1 cells exposed to 10 µg/ml gADIPOQ and 10 µg/ml ox-LDL (n = 6), 1 µg/ml gADIPOQ and 25 µg/ml ox-LDL (n = 6) or 10 µg/ml gADIPOQ and 25 µg/ml ox-LDL (n = 6). Data are expressed as means ± SEM. ** P <0.01 compared with THP-1 cells exposed to 10 µg/ml gADIPOQ and 10 µg/ml ox-LDL; $ P <0.05 and $$ P <0.01 compared with THP-1 cells exposed to 1 µg/ml gADIPOQ and 25 µg/ml ox-LDL. Abbreviations: gADIPOQ, globular adiponectin, iROS, intracellular ROS; mROS, mitochondrial ROS; ox-LDL, oxidized LDL; ROS, reactive oxygen species.

Article Snippet: Furthermore, cDNA clones (OriGene) for IRAK3 (TNFAIP3 and SOD2) were used to double check the primer specificity.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: PLoS ONE

Article Title: Interleukin-1 Receptor-Associated Kinase-3 Is a Key Inhibitor of Inflammation in Obesity and Metabolic Syndrome

doi: 10.1371/journal.pone.0030414

Figure Lengend Snippet: Gene expression was analyzed using qRT-PCR and mROS production was determined by flow cytometry in THP-1 cells exposed to ( A ) 5.5 mM D-glucose and 9.5 mM D-mannitol (osmotic control) or 15 mM D-glucose (n = 6), and ( B ) 100 ng/ml IL-6 (n = 6). Data shown are means ± SEM. * P <0.05, ** P <0.01 and *** P <0.001 compared with THP-1 cells exposed to 5.5 mM D-glucose or PBS vehicle. ( C ) Gene/protein expression and ROS production in THP-1 cells transiently transfected with siRNA targeting IRAK3 (n = 10) or in THP-1 cells exposed to 100 mU/ml glucose oxidase with (n = 4) or without (n = 5) silencing of IRAK3 . Data shown are means ± SEM. * P <0.05, ** P <0.01 and *** P <0.001 compared with THP-1 control cells or THP-1 cells transfected with negative control siRNA; $$ P <0.01 and $$$ P <0.001 compared with THP-1 cells transfected with IRAK3 siRNA; ## P <0.01 and ### P <0.001 compared with THP-1 cells exposed to glucose oxidase. Abbreviations: iROS, intracellular ROS; mROS, mitochondrial ROS; ROS, reactive oxygen species.

Article Snippet: Furthermore, cDNA clones (OriGene) for IRAK3 (TNFAIP3 and SOD2) were used to double check the primer specificity.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Transfection, Negative Control