quantitative pcr experiments  (MiniPCR Inc)

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: OPTN expression is downregulated in AD patients and APP/PS1 transgenic mice. A – D Transcriptome data of the entorhinal cortex, hippocampus, frontal cortex, and temporal cortex in AD patients were analyzed after normalization. E – H Nine-month-old APP/PS1 transgenic mice were anesthetized and euthanized to obtain the cerebral cortex and hippocampus. E Expression of OPTN in the cerebral cortex and hippocampus of APP/PS1 transgenic mice was detected by qRT-PCR using GAPDH as an internal control. F The protein level of OPTN in the cerebral cortex and hippocampus of APP/PS1 transgenic mice was assessed by western blotting using β-actin as an internal control. G ImageJ software was used to semiquantitatively analyze the fold change of OPTN relative to β-actin. H WT or APP/PS1 Tg mice were double-stained for Iba1 (green) and OPTN (red). I Expression of OPTN in microglia in the cortex and hippocampus of APP/PS1 transgenic mice at 9 months of age was detected by flow cytometry. The data represent the means ± S.E. of independent experiments. APP/PS1 transgenic mice were compared with WT mice * P < 0.05, ** P < 0.01

Article Snippet: All reagents for the quantitative real-time PCR (qRT-PCR) experiments were purchased from Bio-Rad Laboratories (Hercules, CA, USA).

Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR, Western Blot, Software, Staining, Flow Cytometry

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: The AIM2 inflammasome is activated in AD patients and APP/PS1 transgenic mice. A – D Brain transcriptome data from patients with AD and controls were collected from the GEO database and normalized for analysis. E – H APP/PS1 transgenic mice at the age of 9 months were anesthetized and euthanized to obtain the cerebral cortex and hippocampus. E Detection of the expression levels of AIM2, ASC, pro-caspase-1, caspase-1 and IL-1β in the cerebral cortex by western blotting. β-actin served as the internal control. In the right panel, ImageJ software was used to semiquantitatively analyze the western blotting results. F qRT-PCR was used to detect the mRNA expression of AIM2 and ASC in the cerebral cortex. GAPDH served as internal control. G The expression of AIM2, ASC, pro-caspase-1, caspase-1 and IL-1β in the hippocampus was detected by western blotting. β-actin served as the internal control. H The mRNA expression of AIM2 and ASC in the hippocampus was detected by qRT-PCR. GAPDH was used as the internal control. The data present means ± S.E. of independent experiment. APP/PS1 transgenic mice were compared with WT mice * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: All reagents for the quantitative real-time PCR (qRT-PCR) experiments were purchased from Bio-Rad Laboratories (Hercules, CA, USA).

Techniques: Transgenic Assay, Expressing, Western Blot, Software, Quantitative RT-PCR

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: Aβo activates the AIM2 inflammasome. A – D BV2 cells were treated with Aβo for 12 h. A Expression levels of AIM2, ASC, pro-caspase-1 and caspase-1 were detected by western blotting. β-actin served as an internal control. B ImageJ software was used for semiquantitative analysis of western blots. C qRT-PCR was used to detect the mRNA expression of AIM2 and ASC with GAPDH as an internal control. D Secretion of IL-1β was evaluated by ELISA. E – H Primary microglia were treated with Aβo for 12 h. E Protein levels of AIM2, ASC, pro-caspase-1, and caspase-1 were detected by western blotting with β-actin as the internal control. F ImageJ software was used to semiquantitatively analyze the fold change in AIM2, ASC, pro-caspase-1 and caspase-1 relative to β-actin. G mRNA expression of AIM2 and ASC was detected by qRT-PCR with GAPDH as an internal control. H Secretion of IL-1β was detected by ELISA. The data present means ± S.M. of independent experiment. Aβo treatment were compared with vehicle treatment * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: All reagents for the quantitative real-time PCR (qRT-PCR) experiments were purchased from Bio-Rad Laboratories (Hercules, CA, USA).

Techniques: Expressing, Western Blot, Software, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

doi: 10.1186/s12974-021-02327-4

Figure Lengend Snippet: OPTN negatively regulates RIPK1 inflammatory signaling pathways. A BV2 cells with OPTN silenced were treated with Aβo for 12 h. Then, OPTN, RIPK1, p-IκBα, and IκBα in the cytoplasm and NF-κB in the cytoplasm or nucleus were detected by western blot with β-actin as an internal control. B – G ImageJ software was used to semiquantitatively analyze the optical density of western blots. H – K OPTN-silenced BV2 cells were treated with Aβo for 12 h. H OPTN mRNA expression was detected by qRT-PCR using GAPDH as an internal control. I RIPK1 RNA expression was detected by qRT-PCR using GAPDH as an internal control. J Extracellular secretion of IL-1β was assessed by ELISA. K The binding activity of NF-κB was evaluated by dual-luciferase assay. The data are presented as the means ± S.M. of independent experiment. OPTN-silenced BV2 cells compared to control BV2 cells or Aβo-treated BV2 cells compared to vehicle BV2 cells, *P < 0.05, ** P < 0.01, *** P < 0.001. L – V BV2 cells with ectopic overexpression of OPTN in the absence or presence of Aβo treatment for 12 h. L Protein levels of OPTN, RIPK1, p-IκBα, and IκBα in the cytoplasm and NF-κB in the cytoplasm or nucleus were detected by western blot using β-actin as an internal control. N – R ImageJ software was used to semiquantitatively analyze the western blot results. S mRNA expression of OPTN was detected by qRT-PCR using GAPDH as an internal control. T mRNA expression of RIPK1 was detected by qRT-PCR using GAPDH as an internal control. U Extracellular secretion of IL-1β was assessed by ELISA. V The binding activity of NF-κB was evaluated using a dual-luciferase assay. The data present means ± S.M. of independent experiment. OPTN overexpressed BV2 cells compared with control BV2 cells or Aβo-treated BV2 cells compared with vehicle BV2 cells, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: All reagents for the quantitative real-time PCR (qRT-PCR) experiments were purchased from Bio-Rad Laboratories (Hercules, CA, USA).

Techniques: Western Blot, Software, Expressing, Quantitative RT-PCR, RNA Expression, Enzyme-linked Immunosorbent Assay, Binding Assay, Activity Assay, Luciferase, Over Expression