taqman primer/probe set human gapdh  (Thermo Fisher)

Journal: International Journal of Molecular Sciences

Article Title: Inhibitory Effect of a Human MicroRNA, miR-6133-5p, on the Fibrotic Activity of Hepatic Stellate Cells in Culture

doi: 10.3390/ijms21197251

Figure Lengend Snippet: The impact of miR-6133 target genes on the anti-fibrotic activity of LX-2 cells. ( A ) RNAseq analysis revealed that 373 genes were downregulated in miR-6133-treated LX-2 cells compared with those treated with miControl, with statistical significance ( p < 0.05). Thirty-six of these genes were also among 438 putative miR-6133-5p target genes predicted by TargetScanHuman v7.2. The levels of TGFBR2 (which encodes Transforming Growth Factor Beta Receptor 2) in the miR-6133-treated and miControl-treated LX-2 cells ( n = 3) were determined by RT-qPCR. ( B ) LX-2 cells were transfected with small interfering RNAs (siRNAs) corresponding to each of the 36 genes or a non-targeting control siRNA (siControl) at a final concentration of 20 nM, 24 h before treatment with rhTGF-β1 (5 ng/mL). The expression of COL1A1 and ACTA2 was determined by RT-qPCR. Fold change was calculated as the ratio over the expression in the siControl-treated sample (indicated by a dotted line). Error bars represent means ± standard deviations ( n = 3). * p < 0.05; ** p < 0.01.

Article Snippet: The primer–probe sets for RT-qPCR analysis of human COL1A1 , ACTA2 , TGFBR2 , FGFR1 , and GAPDH genes were purchased from Thermo Fisher Scientific.

Techniques: Activity Assay, Quantitative RT-PCR, Transfection, Control, Concentration Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibitory Effect of a Human MicroRNA, miR-6133-5p, on the Fibrotic Activity of Hepatic Stellate Cells in Culture

doi: 10.3390/ijms21197251

Figure Lengend Snippet: The effect of knockdown of TGFBR2 and FGFR1 (which encodes Fibroblast Growth Factor Receptor 1), on the activation of intracellular signaling pathways. ( A ) LX-2 cells were treated with an TGFBR2 siRNA or siControl at a final concentration of 20 nM, 24 h before treatment with rhTGF-β1 (5 ng/mL). Protein extracts were prepared from the samples collected 24 h after rhTGF-β1 treatment and subjected to Western blot analysis. ( B ) LX-2 cells were treated with an FGFR1 siRNA, TGFBR2 siRNA, or siControl at a final concentration of 20 nM, 24 h before treatment with rhTGF-β1. The lysates were analyzed by Western blot analysis. ( C ) Values of fragments per kilobase of exon per million reads mapped (FPKM) for FGF1 , FGF2 , FGF5 , FGFR1 , HBEGF , VEGFA , IRS1 , PIK3CD , and PIK3R2 genes, deduced from the RNAseq analysis comparing samples treated with miR-6133-5p and miControl. Error bars represent means ± standard deviations ( n = 3). * p < 0.05; ** p < 0.01.

Article Snippet: The primer–probe sets for RT-qPCR analysis of human COL1A1 , ACTA2 , TGFBR2 , FGFR1 , and GAPDH genes were purchased from Thermo Fisher Scientific.

Techniques: Knockdown, Activation Assay, Protein-Protein interactions, Concentration Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Inhibitory Effect of a Human MicroRNA, miR-6133-5p, on the Fibrotic Activity of Hepatic Stellate Cells in Culture

doi: 10.3390/ijms21197251

Figure Lengend Snippet: Schema of anti-fibrotic mechanisms via miR-6133-5p. TGF-β signaling mediated by the receptor TGFBR2 and its receivers, Smad2/3, control, COL1A1 , and ACTA2 . miR-6133-5p directly suppresses the expression of TGFBR2 . miR-6133-5p decreases phosphorylation (activation) of Akt, ERK, and JNK, possibly by targeting FGFR1 which results in the suppression of FGF/FGFR axis. Akt and JNK regulate COL1A1 and ACTA2 expression.

Article Snippet: The primer–probe sets for RT-qPCR analysis of human COL1A1 , ACTA2 , TGFBR2 , FGFR1 , and GAPDH genes were purchased from Thermo Fisher Scientific.

Techniques: Control, Expressing, Phospho-proteomics, Activation Assay

Journal: eLife

Article Title: HIV-1 Vpu is a potent transcriptional suppressor of NF-κB-elicited antiviral immune responses

doi: 10.7554/eLife.41930

Figure Lengend Snippet:

Article Snippet: The maximal amount of RNA was reversely transcribed with the PrimeScript RT Reagent Kit (Perfect Real Time) (TAKARA, Kusatsu, Japan) using Oligo dT primers and random hexamers. cDNA was subjected to quantitative real time PCR using primer/probe sets for human IFNB1 and GAPDH (ThermoFisher Scientific, Waltham, MA) in singleplex reactions.

Techniques: Derivative Assay, Purification, Recombinant, Plasmid Preparation, Mutagenesis, Luciferase, Virus, Sequencing, Gene Expression, Control, Ab Array, Sample Prep, Western Blot, Enzyme-linked Immunosorbent Assay, Software