quantitative real-time pcr experiment  (Sangon Biotech)

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

Journal: BMC Evolutionary Biology

Article Title: Comparative analyses of glycerotoxin expression unveil a novel structural organization of the bloodworm venom system

doi: 10.1186/s12862-017-0904-4

Figure Lengend Snippet: Quantitative real-time PCR (qPCR) expression levels (shown as Fold Change, RQ) between biological groups (putative venom glands (pvg), pharyngeal lobes, and posterior body wall) of five analyzed GLTx transcripts (GLTx paralog 1–3, and adjacent gene regions GLTx-3’ and GLTx-5’; for details see Material and methods section “Quantitative real-time PCR”) in G. tridactyla ( n = 10; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05). a Relative GLTx expression (logarithmic scale) in putative venom glands (grey) and pharyngeal lobes (orange) in comparison to the GLTx expression signal exhibited by the body tissue (RQ = 1). Relative GLTx expression in the pharyngeal lobes and putative venom glands is significantly different from the expression signal in the body tissue. b Relative GLTx expression (linear scale) within the pharyngeal lobes in comparison to the putative venom glands (RQ = 1). Relative GLTx expression is significantly different between both putative venom glands and pharyngeal lobes

Article Snippet: Quantitative real-time PCR (qPCR) experiments were performed on Glycera tridactyla Schmarda, 1861 (Annelida, Glyceridae) specimens obtained from the Roscoff marine biological station in February 2015.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Comparison