5× hot firepol evagreen qpcr mix plus (rox)  (Solis BioDyne)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Restoration of myogenesis in ALS-myocytes through miR-26a-5p-mediated Smad4 inhibition and its impact on motor neuron development

doi: 10.1016/j.omtn.2025.102581

Figure Lengend Snippet: miRNA expression (A) Heatmap of differentially expressed miRNAs in the medium derived from the conditions described in the sample names (columns). The expression of each miRNA is indicated as relative to the average of the miRNA in the samples. Biological replicates cluster together and G93A G93A samples cluster separated from WT WT and G93A WT . On the right, it is shown miRNA cluster with a low expression in the G93A G93A sample (blue) and the one with high expression in G93A G93A (violet). # and @ indicate miRNAs associated with striated muscle functions according to target function. (B) Relative expression of miRNAs within the blue cluster according to RT-qPCR experiments. (C) Relative expression of miRNAs within the violet cluster according to RT-qPCR. For both (B and C) ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.0005 calculated according to t test between indicated samples considering unequal variance. SD is indicated.

Article Snippet: The total reaction volume was 10 μL, including 2 μL 5× Hot FirePol EvaGreen qPCR Mix (Solis BioDyne), 0.6 μL of 10 μM left primer, 0.6 μL of 10 μM right primer, 1 μL template (10 ng/μL), and 4.2 μL of water.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Tc BDF6 deficiency compromises intracellular amastigote development and infectivity in Trypanosoma cruzi

doi: 10.1101/2025.07.21.665854

Figure Lengend Snippet: (A) Parasitemia was assessed by direct blood examination at multiple time points post-infection. Only wild-type (Dm28c) parasites exhibited detectable parasitemia following immunosuppression. (B) Quantification of parasite load in blood by qPCR revealed a significant reduction in TcBDF6 −/− mutants compared to WT, while the add-back strain ( TcBDF6 −/− pBDF6) showed partial restoration. (C) Relative spleen weight, used as an indicator of immune activation, was significantly elevated in WT-infected mice but not in those infected with TcBDF6 −/− mutants or the add-back strain. (D, E) Parasite load in heart and muscle tissues, measured by qPCR, was markedly decreased in TcBDF6 −/− infected mice, with partial recovery in the add-back group. (F–J) Infections in immunodeficient BALB/c NUDE mice, which lack functional T cells. (F) Parasitemia kinetics showed robust parasite proliferation in WT-infected nude mice, while TcBDF6 −/− mutants failed to establish significant parasitemia. Complementation partially restored parasitemia levels. (G) qPCR quantification in blood confirmed significantly reduced parasite levels in TcBDF6 −/− -infected BALB/c nude mice, with partial restoration in the add-back strain. (H) Relative spleen weights remained low in TcBDF6 −/− infected mice, comparable to uninfected controls, suggesting reduced immune activation. (I, J) Parasite burden in heart and muscle tissues was significantly reduced in TcBDF6 −/− infected nude mice, with partial restoration upon complementation. Data are expressed as median ± range and were analyzed using non-parametric tests (Kruskal-Wallis followed by Mann-Whitney U test). Results represent two independent experiments with 3–5 animals per group. Statistical comparisons between groups were performed using non-parametric tests. Significance levels are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001 (between specified groups); #p < 0.05, ##p < 0.01 versus all other groups. and show a marked difference in p-values. While one comparison reaches statistical significance (p < 0.05), the other shows a trend toward significance (p = 0.07), suggesting a potential biological effect despite not meeting conventional thresholds. Notably, the comparison appears different visually but is not statistically significant.

Article Snippet: PCR reactions were performed using a HOT FIREPol EvaGreen qPCR Mix Plus kit (Solis Biodyne®) with a StepOneTM Real-Time PCR Systems (Applied Biosystems®) instrument.

Techniques: Infection, Activation Assay, Functional Assay, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: UVA irradiation promotes ROS-mediated formation of the common deletion in mitochondrial DNA

doi: 10.1101/2025.07.09.663484

Figure Lengend Snippet: (a) Schematic representations of the UVA (left) and UVB (right) exposures performed on the BJ-5ta human fibroblast cell line. Cells in medium devoid of FBS and antibiotics were irradiated with UV twice per day with the indicated doses of UVA (red bars) or UVB (cyan bars). During recovery intervals (grey bars) cells were placed in an incubator with medium containing FBS and antibiotics. Harvesting was performed 30 minutes after the last indicated irradiation. (b) Relative quantification of wild-type mtDNA (mtDNA WT, light bars) and mtDNA harboring the common deletion (mtDNA CD, dark bars) using qPCR in Unexposed and UVA-(left) or UVB-exposed (right) cells. (c) Relative survival of BJ-5ta cells irradiated with indicated doses of UVA (left) or UVB (right) compared to Unexposed cells (grey dotted line). ( d) Citrate synthase activity in UVA-(top) and UVB-exposed (bottom) BJ-5ta cells. The citrate synthase activity in UVA and UVB exposed cells is expressed as fold-change per well to Unexposed control cells (grey dotted line). (e) Oxygen consumption rates (OCR) at basal and stimulated (maximal) ETC activity in UVA (top) and UVB (bottom) exposed cells. (f) Extracellular acidification rates (ECAR) at basal and maximal ETC activity in UVA-(top) and UVB-(bottom) exposed cells. For (b-d) n = 3 biological replicates were used and (c-d) and for (e-f) n = 7 biological replicates. Error bars represent standard deviation.

Article Snippet: For each sample 4-6 ng cDNA, 6 μL HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne), 0.4 μM forward primer, 0.4 μM reverse primer, and nuclease-free water were prepared to a total volume of 12 μL.

Techniques: Irradiation, Quantitative Proteomics, Activity Assay, Control, Standard Deviation

Journal: bioRxiv

Article Title: UVA irradiation promotes ROS-mediated formation of the common deletion in mitochondrial DNA

doi: 10.1101/2025.07.09.663484

Figure Lengend Snippet: (a) Schematic representation of the UVA and antioxidants (AO) co-treatments performed on the BJ-5ta human fibroblasts cell line. Cells were pre-incubated with AO for 1 h in complete medium (purple bar). The AO-containing medium was removed, and cells were irradiated twice with UVA 10 J/cm 2 in medium devoid of FBS and antibiotics (red bars). In between irradiations and after the last irradiation cells were incubated with AO for 2 h and 30 minutes, respectively. Harvesting (dotted arrows) was performed 30 minutes before and after the first last AO incubation, assessing the ROS content at the start (S) or end (E) of the exposure regime. Sampling for qPCR-based assessment of the mtDNA CD content is also indicated. (b) Cellular ROS content in BJ-5ta under-going the co-treatment with reduced glutathione (GSH, left), Coenzyme Q10 (CoQ10, right) and/or the corresponding vehicles H2O or Dimethylformamide (DMF). Cells were collected at the start and at the end of the UV exposure procedure and the ROS content was quantified using the DCF assay. Data are expressed as fold change to cells exposed to vehicle at the end of the first day. (c) Relative quantification of wild-type mtDNA (mtDNA WT, light bars) and mtDNA harboring the common deletion (mtDNA CD, dark bars) by qPCR of BJ-5ta cells exposed to UVA and co-treated with glutathione (GSH, left), Coenzyme Q10 (CoQ10, right) and/or the corresponding vehicles H 2 O or Dimethylformamide (DMF) as in the scheme in . Left panel: n = 3 and right panel n = 2. (d) Basal levels of ROS in BJ-5ta cells and human fibroblasts from a Kearns-Sayre syndrome (KSS) patient displaying inherent levels of oxidative stress. Fold change of cellular ROS content expressed as fold-change to ROS content in BJ-5ta cells (grey dotted line). (e) Schematic representation of the AO treatments in KSS cells. Cells were acutely exposed to AO for 4 h per day in complete medium, for 2 days. Cells were harvested 30 mins after the last incubation and their ROS and mtDNA CD contents were assessed by DCF and qPCR assays, respectively. (f) ROS content in KSS cells treated with vehicle or with GSH or CoQ10 for 4 h per day over 2 days. (g) Relative quantification of mtDNA WT (light bars) and mtDNA CD (dark bars) by qPCR in KSS cells treated with vehicle or with the indicated AO for 8 h, 4 h per day. n = 3 biological replicated for panel (b-g) . Error bars represent standard deviations.

Article Snippet: For each sample 4-6 ng cDNA, 6 μL HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne), 0.4 μM forward primer, 0.4 μM reverse primer, and nuclease-free water were prepared to a total volume of 12 μL.

Techniques: Incubation, Irradiation, Sampling, DCF Assay, Quantitative Proteomics

Journal: bioRxiv

Article Title: UVA irradiation promotes ROS-mediated formation of the common deletion in mitochondrial DNA

doi: 10.1101/2025.07.09.663484

Figure Lengend Snippet: (a) Schematic representations of the UVA exposure regime performed on three-dimensional full-thickness human skin equivalents (HSE). HSE were irradiated twice per day with the indicated doses of UVA (red bars), interrupted by recovery intervals (grey bars). Harvesting was performed 30 minutes after the last indicated irradiation. Full-thickness HSE (inlet) are composed of primary human fibroblasts and keratinocytes, constituting the dermis (D) and epidermis (E) respectively, seeded on a plastic air-lifted scaffold. Cellular proliferation and interactions between the two skin layers lead to formation of the corneum layer (C), reconstituting the structure and functionality of human skin. (b) Relative quantification of wild-type mtDNA (mtDNA WT, light bars) and mtDNA harboring the common deletion (mtDNA CD, dark bars) by qPCR in HSE exposed to increasing doses of UVA. Dermis (left) and Epidermis (left) were analyzed separately. (c) Representative microscopy images of hematoxylin-eosin-stained HSE sections, in unexposed conditions or exposed to the indicated doses of UVA. (d) Left: dot blots performed with mtDNA extracted from the epidermis and dermis of HSE, untreated or treated with 100 J/cm 2 UVA and hybridized with anti-8oxodG and anti-dsDNA antibodies. Right: quantitation of n=3 biological replicates of dot blots. (e) Schematic representation of the UVA and CoQ10 co-treatments performed on HSE. Cells were pre-incubated for 1 h with 5 µM CoQ10 in complete medium (purple bar). The CoQ10-containing medium was removed, and HSE were irradiated twice with UVA 10 J/cm 2 . In between irradiation and after the last irradiation cells were incubated with CoQ10 for 30 minutes, respectively. Harvesting (dotted arrows) was performed 30 minutes after removing the CoQ10-containing medium. (f) Relative quantification of mtDNA WT (light bars) and mtDNA CD (dark bars) by qPCR of HSE exposed to UVA and co-treated 5 µM CoQ10 and/or the corresponding vehicle scheme in . Dermis (left) and Epidermis (left) were analyzed separately. n = 3 biological replicated for panel (b-g) Error bars represent standard deviations.

Article Snippet: For each sample 4-6 ng cDNA, 6 μL HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne), 0.4 μM forward primer, 0.4 μM reverse primer, and nuclease-free water were prepared to a total volume of 12 μL.

Techniques: Irradiation, Quantitative Proteomics, Microscopy, Staining, Quantitation Assay, Incubation

Journal: bioRxiv

Article Title: UVA irradiation promotes ROS-mediated formation of the common deletion in mitochondrial DNA

doi: 10.1101/2025.07.09.663484

Figure Lengend Snippet: (a) Fold-change of cellular ROS content in BJ-5ta cells quantified by measuring the fluorescent 2’, 7’ –dichlorofluorescein (DCF) in UVA-(left) and UVB-exposed (right) cells to unexposed cells (grey dotted line). n = 3 biological replicates and analyzed by paired t-test comparing to unexposed control. (b) Representative dot blots (DB, top) and relative DBs quantitation (bottom) in fold change compared to unexposed cells (dotted grey line). DBs were performed on mtDNA extracted from BJ-5ta cells irradiated with the indicated doses of UVA (left) or UVB (right). Hybridization was performed with an anti-8-oxo-dG, an anti-cyclobutane pyrimidine dimer (CPDs), and an anti-double stranded DNA (dsDNA) antibody as a loading control. (c) Primer extension assays with mitochondrial DNA Pol γ replication past 40mer templates containing either an unmodified G Template (left) or a modified 8-oxo-dG template (right). Templates were pre-annealed opposite 25mer primers containing a 5’ Cy3 dye. (d) BJ-5ta cells were exposed to the indicated doses of UVA (left) and UVB (right) according to the scheme in . mtDNA samples were collected every 24 h for seven days after exposure in absence of further stimuli. Relative quantification of wild-type mtDNA (mtDNA WT, dark bars) and mtDNA harboring the common deletion (mtDNA CD, light bars) by qPCR in unexposed and UVA-(left) or UVB-exposed (right) cells. (e) Representative DBs (left) and relative DBs quantitation (right) in fold change compared to unexposed cells (dotted grey line). DBs were performed on mtDNA extracted from BJ-5ta cells irradiated with the indicated doses of UVA (top) or UVB (bottom) and collected every 24 h for seven days after exposure in absence of further stimuli. Hybridization was performed with an anti-8-oxo-dG, an anti-cyclobutane pyrimidine dimer (CPDs), and an anti-double stranded DNA (dsDNA) antibody as a loading control. n = 3 biological replicates for (b, d-e). Error bars represent standard deviations.

Article Snippet: For each sample 4-6 ng cDNA, 6 μL HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne), 0.4 μM forward primer, 0.4 μM reverse primer, and nuclease-free water were prepared to a total volume of 12 μL.

Techniques: Control, Quantitation Assay, Irradiation, Hybridization, Modification, Quantitative Proteomics

Journal: bioRxiv

Article Title: UVA irradiation promotes ROS-mediated formation of the common deletion in mitochondrial DNA

doi: 10.1101/2025.07.09.663484

Figure Lengend Snippet: (a) RNA-seq results depicting the genes most affected in their expression upon exposure of BJ-5ta cells with UVA 100 J/cm 2 according to the scheme in . 2000 most up- and downregulated genes were binned in 6 clusters (colored bars, left) and GEO terms were assigned to each cluster (right). (b) RNA-seq analysis of genes involved in mtDNA maintenance (mtDNA replication, repair and degradation) in unexposed and UVA-exposed BJ5-ta fibroblasts. (c) qPCR assays for expression of mtDNA maintenance genes upon UVA-exposed cells. n = 3 biological replicates were used for each graph. Statistical analysis was performed with a paired t-test comparing UV-exposed samples to matched unexposed control. (d) RNA-seq analysis of mtDNA-encoded genes in five unexposed and UVA-treated biological replicates. (e) qPCR analysis for the expression of mtDNA-encoded genes in UVA-exposed cells, expressed as fold change over unexposed cells (grey dotted line). Color codes indicate the ETC complexes to which the encoded proteins belong to, rRNAs and the Humanin micropeptide. n = 3 biological replicates were used for each graph. Statistical analysis was performed with a paired t-test comparing UV-exposed samples to matched unexposed control.

Article Snippet: For each sample 4-6 ng cDNA, 6 μL HOT FIREPol EvaGreen qPCR Mix (Solis Biodyne), 0.4 μM forward primer, 0.4 μM reverse primer, and nuclease-free water were prepared to a total volume of 12 μL.

Techniques: RNA Sequencing, Expressing, Control