real time quantitative reverse transcription pcr rt qpcr reactions  (Roche)

 
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    Roche real time quantitative reverse transcription pcr rt qpcr reactions
    Quantitative <t>RT-PCR</t> analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the <t>RT-qPCR</t> analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).
    Real Time Quantitative Reverse Transcription Pcr Rt Qpcr Reactions, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection"

    Article Title: Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150711

    Quantitative RT-PCR analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the RT-qPCR analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).
    Figure Legend Snippet: Quantitative RT-PCR analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the RT-qPCR analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).

    Techniques Used: Quantitative RT-PCR, Infection, Microarray

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection
    Article Snippet: .. Three technical replicates of each three biological replicates (for infected tuber tissue only two biological replicates) were used in subsequent Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) reactions using FastStart Universal Probe Master with Rox (Roche Diagnostics, Switzerland). ..

    Infection:

    Article Title: Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection
    Article Snippet: .. Three technical replicates of each three biological replicates (for infected tuber tissue only two biological replicates) were used in subsequent Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) reactions using FastStart Universal Probe Master with Rox (Roche Diagnostics, Switzerland). ..

    Quantitative RT-PCR:

    Article Title: Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection
    Article Snippet: .. Three technical replicates of each three biological replicates (for infected tuber tissue only two biological replicates) were used in subsequent Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) reactions using FastStart Universal Probe Master with Rox (Roche Diagnostics, Switzerland). ..

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    Roche rna
    Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si <t>RNA</t> ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative <t>PCR</t> using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P
    Rna, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche gene expression real time quantitative reverse transcription pcr qrt pcr analyses
    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by <t>qRT-PCR</t> and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.
    Gene Expression Real Time Quantitative Reverse Transcription Pcr Qrt Pcr Analyses, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche negative strand hcv pcr amplifications
    Effect of increasing concentrations of IFN-α on the accumulation of positive- and negative-strand <t>HCV</t> RNA in primary hepatocyte cultures infected in vitro. Cultures FT147 (infected with serum S26) and FT161 (infected with serum S42) were treated for 5 and 8 days with IFN-α concentrations ranging from 1,000 to 10,000 U/ml and 500 to 5,000 U/ml, respectively. Qualitative detection of positive-sense (+) and negative-sense (-) HCV RNA strands is shown in cultures FT147 (a) and FT161 (b). In both instances, positive-strand HCV RNA was detected at all concentrations used, whereas the negative strand was never detected. MW, molecular size standards. (c) In culture FT161, LightCycler real-time <t>RT-PCR</t> quantitative analysis of the same extracts showed a reduction in the amount of positive-sense HCV RNA strand in the culture when the IFN-α concentration increased, suggesting IFN-α concentration-dependent inhibition of HCV replication in the culture. Similar results (not shown) were obtained with culture FT187 infected with serum S155.
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    Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si RNA ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative PCR using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P

    Journal: Cancer Science

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1

    doi: 10.1111/cas.13754

    Figure Lengend Snippet: Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si RNA ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative PCR using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P

    Article Snippet: 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Construct, Chromatin Immunoprecipitation, Over Expression, Activity Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

    T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

    Article Snippet: Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche).

    Techniques: Activation Assay, Transformation Assay, Purification, Selection, RNA Sequencing Assay, Chromatin Immunoprecipitation, Sequencing, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Activity Assay, In Vitro, Expressing, Quantitative RT-PCR, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Positive Control, Staining, FACS

    Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Expression of potential ripening-related transcription factors (TFs) in response to post-harvest ABA and sugar treatments (A) and during bilberry fruit development (B) . The gene expression was analyzed 4 days after the beginning of the treatments. The treatments were: ABA (0.5 and 2 mM), glucose (200 mM), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values in (A) represent means ± SEs of three replicates and asterisks significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Values in (B) represent means ± SEs of four replicates and asterisks significant increase from previous developmental stage in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001). Stages 1–5 indicate the bilberry fruit developmental stages from flower to ripe berry.

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of post-harvest ABA and sugar treatments on the expression of key ABA and sucrose biosynthetic genes VmNCED1 (A) , VmSS (B) , VmSPS1 (C) , VmSPS2 (D) , and VmSPS3 (E) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of post-harvest ABA and sugar treatments on the expression of key ABA and sucrose biosynthetic genes VmNCED1 (A) , VmSS (B) , VmSPS1 (C) , VmSPS2 (D) , and VmSPS3 (E) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of post-harvest ABA and sugar treatments on the expression of anthocyanin biosynthetic genes VmCHS (A) , VmCHI (B) , VmF3H (C) , VmF3 ′ H (D) , VmF3 ′ 5 ′ H (E) , VmDFR (F) , VmANS (G) , and VmUFGT (H) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of post-harvest ABA and sugar treatments on the expression of anthocyanin biosynthetic genes VmCHS (A) , VmCHI (B) , VmF3H (C) , VmF3 ′ H (D) , VmF3 ′ 5 ′ H (E) , VmDFR (F) , VmANS (G) , and VmUFGT (H) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (50 and 200 mM), fructose (50 and 200 mM), sucrose (50 and 200 mM), 0.5 mM ABA + 200 mM sucrose, 200 μM fluridone or water (control). Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of three replicates. Asterisks indicate significant differences from respective control ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, one-way ANOVA with Tukey’s HSD test).

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of VmNCED1 silencing on anthocyanin biosynthesis in ripening bilberry fruit. Green unripe fruits still attached to the bilberry plants were injected with VmNCED1 -VIGS vector or pBINTRA6 vector only (control). Arrows indicate injection sites. Fruits were evaluated 4 weeks after injection for color (A) , and the expression of VmNCED1 and the key anthocyanin biosynthetic genes in intact fruits as well as in green and red sectors of chimeric fruits (B) . Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SDs of three replicates.

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of VmNCED1 silencing on anthocyanin biosynthesis in ripening bilberry fruit. Green unripe fruits still attached to the bilberry plants were injected with VmNCED1 -VIGS vector or pBINTRA6 vector only (control). Arrows indicate injection sites. Fruits were evaluated 4 weeks after injection for color (A) , and the expression of VmNCED1 and the key anthocyanin biosynthetic genes in intact fruits as well as in green and red sectors of chimeric fruits (B) . Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SDs of three replicates.

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Injection, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Effect of pre-harvest treatment with ABA on bilberry fruit color (A) , anthocyanin content (B) , and expression of anthocyanin biosynthetic genes (C) . Unripe green berries attached to plants were sprayed with 0.5 mM ABA, 2 mM ABA or water (control). Fruit color and anthocyanin content was evaluated after 7 days from the beginning of the experiment. Total anthocyanin content is expressed as milligrams of cyanidin-3-glucoside equivalents g -1 FW. Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of four replicates. Asterisks indicate significant differences from control in Student’s t -Test ( P ≤ 0.05).

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of pre-harvest treatment with ABA on bilberry fruit color (A) , anthocyanin content (B) , and expression of anthocyanin biosynthetic genes (C) . Unripe green berries attached to plants were sprayed with 0.5 mM ABA, 2 mM ABA or water (control). Fruit color and anthocyanin content was evaluated after 7 days from the beginning of the experiment. Total anthocyanin content is expressed as milligrams of cyanidin-3-glucoside equivalents g -1 FW. Relative expression of the genes was quantified by qRT-PCR and normalized to VmGAPDH . Values represent means ± SEs of four replicates. Asterisks indicate significant differences from control in Student’s t -Test ( P ≤ 0.05).

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of post-harvest ABA and sugar treatments on the expression cell wall modifying genes VmPE1 (A) , VmPE2 (B) , VmPL (C) , VmPG1 (D) , VmPG2 (E) , VmRGLyase (F) , Vm β GAL1 (G) , Vm β GAL2 (H) , VmXTH (I) , VmCEL (J) , VmXYL (K) , VmEXP1 (L) , VmEXP2 (M) , and VmEXP3 (N) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (200), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR after 4 days of the beginning of the experiment and normalized to VmGAPDH . Values represent means ± SEs of three replicates. PE , pectin esterase; PL , pectate lyase; PG , polygalacturonase; RGLyase , rhamnogalacturonate lyase; β GAL , β-galactosidase; XTH , xyloglucan endotransglycosylase/hydrolase; CEL , endo-β - 1,4 glucanase: XYL , β-xylosidase; EXP , expansin. Asterisks indicate significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001).

    Journal: Frontiers in Plant Science

    Article Title: Abscisic Acid Regulates Anthocyanin Biosynthesis and Gene Expression Associated With Cell Wall Modification in Ripening Bilberry (Vaccinium myrtillus L.) Fruits

    doi: 10.3389/fpls.2018.01259

    Figure Lengend Snippet: Effect of post-harvest ABA and sugar treatments on the expression cell wall modifying genes VmPE1 (A) , VmPE2 (B) , VmPL (C) , VmPG1 (D) , VmPG2 (E) , VmRGLyase (F) , Vm β GAL1 (G) , Vm β GAL2 (H) , VmXTH (I) , VmCEL (J) , VmXYL (K) , VmEXP1 (L) , VmEXP2 (M) , and VmEXP3 (N) in bilberry fruit. The treatments were: ABA (0.5 and 2 mM), glucose (200), fructose (200 mM), sucrose (200 mM), 0.5 mM ABA + 200 mM sucrose, or water (control). Relative expression of the genes was quantified by qRT-PCR after 4 days of the beginning of the experiment and normalized to VmGAPDH . Values represent means ± SEs of three replicates. PE , pectin esterase; PL , pectate lyase; PG , polygalacturonase; RGLyase , rhamnogalacturonate lyase; β GAL , β-galactosidase; XTH , xyloglucan endotransglycosylase/hydrolase; CEL , endo-β - 1,4 glucanase: XYL , β-xylosidase; EXP , expansin. Asterisks indicate significant differences from control in Student’s t -Test ( ∗ P ≤ 0.05, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001).

    Article Snippet: Relative Quantification of Gene Expression Real-time quantitative reverse transcription PCR (qRT-PCR) analyses were performed with a LightCycler 480 instrument and software (Roche Applied Sciences, Indianapolis, IN, United States).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of increasing concentrations of IFN-α on the accumulation of positive- and negative-strand HCV RNA in primary hepatocyte cultures infected in vitro. Cultures FT147 (infected with serum S26) and FT161 (infected with serum S42) were treated for 5 and 8 days with IFN-α concentrations ranging from 1,000 to 10,000 U/ml and 500 to 5,000 U/ml, respectively. Qualitative detection of positive-sense (+) and negative-sense (-) HCV RNA strands is shown in cultures FT147 (a) and FT161 (b). In both instances, positive-strand HCV RNA was detected at all concentrations used, whereas the negative strand was never detected. MW, molecular size standards. (c) In culture FT161, LightCycler real-time RT-PCR quantitative analysis of the same extracts showed a reduction in the amount of positive-sense HCV RNA strand in the culture when the IFN-α concentration increased, suggesting IFN-α concentration-dependent inhibition of HCV replication in the culture. Similar results (not shown) were obtained with culture FT187 infected with serum S155.

    Journal: Journal of Virology

    Article Title: Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

    doi: 10.1128/JVI.76.16.8189-8199.2002

    Figure Lengend Snippet: Effect of increasing concentrations of IFN-α on the accumulation of positive- and negative-strand HCV RNA in primary hepatocyte cultures infected in vitro. Cultures FT147 (infected with serum S26) and FT161 (infected with serum S42) were treated for 5 and 8 days with IFN-α concentrations ranging from 1,000 to 10,000 U/ml and 500 to 5,000 U/ml, respectively. Qualitative detection of positive-sense (+) and negative-sense (-) HCV RNA strands is shown in cultures FT147 (a) and FT161 (b). In both instances, positive-strand HCV RNA was detected at all concentrations used, whereas the negative strand was never detected. MW, molecular size standards. (c) In culture FT161, LightCycler real-time RT-PCR quantitative analysis of the same extracts showed a reduction in the amount of positive-sense HCV RNA strand in the culture when the IFN-α concentration increased, suggesting IFN-α concentration-dependent inhibition of HCV replication in the culture. Similar results (not shown) were obtained with culture FT187 infected with serum S155.

    Article Snippet: Positive- and negative-strand HCV PCR amplifications were performed with 3 μl of purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA Master SYBR green (Roche Applied Science) and 0.5 μM (each) HCV primer KY78 and KY80.

    Techniques: Infection, In Vitro, Quantitative RT-PCR, Concentration Assay, Inhibition

    Qualitative assay detection of positive- and negative-strand HCV RNA in a primary culture of healthy human hepatocytes infected in vitro with an HCV-positive serum and effect of IFN-α. The hepatocyte culture FT147, infected 3 days after plating by HCV-positive serum S26, is shown. Positive-strand (+) RNA but not negative-strand (-) RNA was present in the inoculum. (a) Primary hepatocyte culture in the absence of IFN-α. Positive-strand HCV RNA was detected with the qualitative strand-specific r Tth PCR assay from day 1 to the end of the culture (day 12), whereas negative-strand RNA was detected from days 2 to 10. (b) Culture in the presence of 5,000 U of IFN-α per ml. Positive-strand HCV RNA was detected from days 1 to 10, whereas negative-strand RNA was never detected. (c) Culture treated on day 3 with 5,000 U of IFN-α per ml. Positive-strand RNA was detected throughout the culture period, whereas negative-strand RNA was no longer detected after day 5. Similar patterns (not shown) were observed with the following cultures infected with the corresponding sera: FT141 and S23, FT143 and S34, FT144 and S27, FT154 and S23, FT155 and S20, and FT156 and S17. MK, molecular size standards.

    Journal: Journal of Virology

    Article Title: Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

    doi: 10.1128/JVI.76.16.8189-8199.2002

    Figure Lengend Snippet: Qualitative assay detection of positive- and negative-strand HCV RNA in a primary culture of healthy human hepatocytes infected in vitro with an HCV-positive serum and effect of IFN-α. The hepatocyte culture FT147, infected 3 days after plating by HCV-positive serum S26, is shown. Positive-strand (+) RNA but not negative-strand (-) RNA was present in the inoculum. (a) Primary hepatocyte culture in the absence of IFN-α. Positive-strand HCV RNA was detected with the qualitative strand-specific r Tth PCR assay from day 1 to the end of the culture (day 12), whereas negative-strand RNA was detected from days 2 to 10. (b) Culture in the presence of 5,000 U of IFN-α per ml. Positive-strand HCV RNA was detected from days 1 to 10, whereas negative-strand RNA was never detected. (c) Culture treated on day 3 with 5,000 U of IFN-α per ml. Positive-strand RNA was detected throughout the culture period, whereas negative-strand RNA was no longer detected after day 5. Similar patterns (not shown) were observed with the following cultures infected with the corresponding sera: FT141 and S23, FT143 and S34, FT144 and S27, FT154 and S23, FT155 and S20, and FT156 and S17. MK, molecular size standards.

    Article Snippet: Positive- and negative-strand HCV PCR amplifications were performed with 3 μl of purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA Master SYBR green (Roche Applied Science) and 0.5 μM (each) HCV primer KY78 and KY80.

    Techniques: Infection, In Vitro, Polymerase Chain Reaction

    Characteristics of the strand-specific HCV RNA assays used in this study. (a) Strand specificity of the positive-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of positive-strand (+) HCV RNA (100, 10, 1, and 0.1 fg) and of negative-strand (-) HCV RNA (10, 1, and 0.1 pg) synthesized from an appropriate plasmid were subjected to the r Tth RT-PCR assay. The products were analyzed by agarose gel electrophoresis. (b) Strand specificity of the negative-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of negative-strand HCV RNA (100, 10, 1, and 0.1 fg) and positive-strand HCV RNA (10, 1, and 0.1 pg) synthesized from the same plasmid as for panel a were analyzed by the same procedure. (c) Range of linear quantification of the quantitative assay based on real-time PCR using the LightCycler technology and SYBR green I dye for detection. The range of linear quantification of the assay was studied by testing 10-fold serial dilutions of synthetic positive- and negative-sense HCV RNA strands after RT at 70°C with the r Tth polymerase. Each point is the mean of three experimental values for each dilution. y is the slope of the linear plots.

    Journal: Journal of Virology

    Article Title: Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

    doi: 10.1128/JVI.76.16.8189-8199.2002

    Figure Lengend Snippet: Characteristics of the strand-specific HCV RNA assays used in this study. (a) Strand specificity of the positive-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of positive-strand (+) HCV RNA (100, 10, 1, and 0.1 fg) and of negative-strand (-) HCV RNA (10, 1, and 0.1 pg) synthesized from an appropriate plasmid were subjected to the r Tth RT-PCR assay. The products were analyzed by agarose gel electrophoresis. (b) Strand specificity of the negative-strand-specific HCV RNA r Tth RT-PCR assay. Decreasing amounts of negative-strand HCV RNA (100, 10, 1, and 0.1 fg) and positive-strand HCV RNA (10, 1, and 0.1 pg) synthesized from the same plasmid as for panel a were analyzed by the same procedure. (c) Range of linear quantification of the quantitative assay based on real-time PCR using the LightCycler technology and SYBR green I dye for detection. The range of linear quantification of the assay was studied by testing 10-fold serial dilutions of synthetic positive- and negative-sense HCV RNA strands after RT at 70°C with the r Tth polymerase. Each point is the mean of three experimental values for each dilution. y is the slope of the linear plots.

    Article Snippet: Positive- and negative-strand HCV PCR amplifications were performed with 3 μl of purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA Master SYBR green (Roche Applied Science) and 0.5 μM (each) HCV primer KY78 and KY80.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Synthesized, Plasmid Preparation, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, SYBR Green Assay

    Accumulation of positive- and negative-strand HCV RNA in hepatocyte cultures FT172 (a), FT189 (b), and FT195 (c), infected with sera S42, S155, and S155, respectively, as measured by the quantitative LightCycler real-time RT-PCR assay. The hepatocyte cultures were infected 3 days after plating. The cells were harvested 30 min and 1, 3, and 5 days after infection for positive-strand (gray) and negative-strand (black) HCV RNA quantification. The amounts of HCV RNA strands are shown as means ± SEMs of three determinations, expressed in numbers of HCV RNA copies per 2 × 10 6 cells, normalized to GAPDH mRNA. Similar results (not shown) were obtained with culture FT168 infected with serum S34.

    Journal: Journal of Virology

    Article Title: Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

    doi: 10.1128/JVI.76.16.8189-8199.2002

    Figure Lengend Snippet: Accumulation of positive- and negative-strand HCV RNA in hepatocyte cultures FT172 (a), FT189 (b), and FT195 (c), infected with sera S42, S155, and S155, respectively, as measured by the quantitative LightCycler real-time RT-PCR assay. The hepatocyte cultures were infected 3 days after plating. The cells were harvested 30 min and 1, 3, and 5 days after infection for positive-strand (gray) and negative-strand (black) HCV RNA quantification. The amounts of HCV RNA strands are shown as means ± SEMs of three determinations, expressed in numbers of HCV RNA copies per 2 × 10 6 cells, normalized to GAPDH mRNA. Similar results (not shown) were obtained with culture FT168 infected with serum S34.

    Article Snippet: Positive- and negative-strand HCV PCR amplifications were performed with 3 μl of purified cDNA in a 10-μl reaction mixture containing 1 μl of LightCycler-FastStart DNA Master SYBR green (Roche Applied Science) and 0.5 μM (each) HCV primer KY78 and KY80.

    Techniques: Infection, Quantitative RT-PCR