real time rt pcr qpcr  (Solis BioDyne)

Journal: Scientific Reports

Article Title: Core regulon of the global anaerobic regulator Anr targets central metabolism functions in Pseudomonas species

doi: 10.1038/s41598-019-45541-0

Figure Lengend Snippet: Characterization of microaerobic growth and Anr expression in Pseudomonas species. ( A ) Growth under low oxygen conditions with and without KNO 3 . ( B ) Expression of anr measured by Real time qPCR. ( C ) Western Blot analysis of Anr. Lines: 1. Purified recombinant Anr protein of P. extremaustralis used as positive control 2: Marker PageRuler prestained protein ladder (Thermo scientific); 3: P. extremaustralis ; 4: anr mutant of P. extremaustralis 16 used as negative control. 5: P. syringae B728a: 6: P. putida KT2440. Original Western-blot assay with different exposure times used to build C are shown in Fig. S4 . In A: microaerobic cultures were carried out in LB medium supplemented or not with KNO 3 in sealed bottles with 1:2 medium to flask volume ratio and low agitation (50 rpm), incubated for 30 h. In B and C: cultures were performed with KNO 3 . Bars represent mean ± SE. *: indicate significant differences P

Article Snippet: After treatment with DNaseI, cDNA was obtained using random hexamers (Promega) and Revert Aid Reverse Transcriptase (ThermoFisher Scientific, Waltham, USA) following the manufacturer’s instructions. qRT PCR was performed using a MyiQ2 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, USA) and Real Time PCR mix (EvaGreen qPCR Mix Plus, no ROX, Solis Biodyne).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Purification, Recombinant, Positive Control, Marker, Mutagenesis, Negative Control, Incubation

Journal: Scientific Reports

Article Title: Gene activation by dCas9-CBP and the SAM system differ in target preference

doi: 10.1038/s41598-019-54179-x

Figure Lengend Snippet: Direct fusion of the CBP HAT domain to dCas9 outperforms a MS2 coat protein-CBP fusion plus dCas9 combination. ( A ) Schematic drawings of dCas9 fused to the HAT domain of CBP, MS2 coat protein fused to the CBP HAT domain (MCP-CBP) where two MCP dimers recognize two MS2 loops in the gRNA and dCas9 thereby brings four CBP domains to the locus, dCas9-VPR where three activation domains are fused to dCas9, and the synergistic activation mediator (SAM) system where MCP targets eight activation domains to dCas9 fused with the VP64 activation domain. ( B ) RT-qPCR showing twist ( twi ) expression in Drosophila S2 cells and in S2 cells transfected with UAS-dCas9 fusions or UAS-dCas9 and UAS-MCP fusions together with Actin -Gal4 in the presence of a control gRNA or twi promoter gRNA. Expression is plotted relative to RP49 . n = 3 biological replicates and error bars represent S.E.M. One-way ANOVA with post hoc Tukey test was used to calculate statistically significant differences. The full statistical analysis and fold activation in relation to the control (QUAS) gRNA is shown in Supplementary Table 2 . The F2161A mutation disrupts the catalytic function of the CBP HAT domain. Below the graph is a schematic drawing of the twi locus. ( C ) Western blot showing expression of the dCas9 and MCP fusion proteins. Uncropped images are shown in Supplemental Fig. S9 .

Article Snippet: The cDNA was used in qPCR with 5X HOT FIREPol Evagreen qPCR Mix Plus (Solis Biodyne) and primers listed in Supplementary Table .

Techniques: HAT Assay, Activation Assay, Quantitative RT-PCR, Expressing, Transfection, Mutagenesis, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages

doi: 10.1186/s12964-018-0229-y

Figure Lengend Snippet: Effect of dynasore hydrate on the SW480 and SW620 EV-induced changes on the expression of the surface marker CD14 and on the gene expression of CXCL10 and IL-10 in M0 macrophages. a Flow cytometry analysis showing the percentage of CD14-positive M0 macrophages. The graphs represent mean ± SD (n = 2). Statistical analysis was carried out with t-test. * p ≤ 0.05, ** p ≤ 0.01 b qPCR analysis showing changes in CXCL10 and IL-10 gene expression (n = 3).

Article Snippet: Real-time RT-PCR was performed for quantitative gene expression analysis using 5× HOT FIREPol EvaGreen qPCR Mix Plus (ROX) according to the manufacturer’s instructions.

Techniques: Expressing, Marker, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-7 Restores Phenotypes in Myotonic Dystrophy Muscle Cells by Repressing Hyperactivated Autophagy

doi: 10.1016/j.omtn.2019.11.012

Figure Lengend Snippet: Impaired Expression of Autophagy-Related Genes Is Restored by Increasing miR-7 Levels in DM1 Myoblasts (A) Quantification of relative expression of autophagy-related genes ( ATG4A , ATG7 , ATG5 , ATG2B , ATG3 , and VPS34 ) in DM1 myoblasts by qRT-PCR using the 2 −ΔΔCt method. Green dashed line indicates the relative expression levels of the genes in CNT myoblasts. Logarithmic representation on base 2 (log 2 ) of the qRT-PCR quantification of (B) ATG4A , (C) ATG7 , (D) ATG5 , (E) ATG2B , (F) ATG3 , and (G) VPS34 in CNT (purple) and DM1 (blue) myoblasts transdifferentiated for 7 days after transfection with the indicated concentration of antagomiR-7 (in control TDMs) or agomiR-7 (in DM1 TDMs), respectively. Gene expression levels were normalized to cells transfected with antagomiR or agomiR scramble at each concentration. In all cases, GAPDH expression was used as reference gene (n = 3). Data were obtained using the 2 −ΔΔCt method. The bar graphs show mean ± SEM. *p

Article Snippet: Specific primers were used to analyze the alternative splicing of SPTAN1 , BIN1 , ATP2A1 , INSR , DMD , cTNT , and DLG1 in CNT and DM1 muscle cells ( ). qRT-PCR was performed using 2 ng of cDNA template with 5× HOT FIREPol EvaGreen qPCR mix plus (ROX) (Solis BioDyne) and QuantiFast probe PCR kit reagent (QIAGEN; Vedbaek, Denmark) and specific primers and probes ( ).

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