power sybr green pcr master mix  (Thermo Fisher)


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    Name:
    Power SYBR Green PCR Master Mix
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    Alternative Product Try PowerUp SYBR Green Master Mix our newest high performance SYBR dye based master mix for superior performance at a very competitive price With PowerUp SYBR Green Master Mix we ve taken the best of Power SYBR Green PCR Master Mix and added additional capabilities for your gene expression analysis Get superior nucleic acid quantitation with Applied Biosystems Power SYBR Green Master Mix Power SYBR Master Mix offers superior sensitivity and reproducibility detecting as few as two copies of a target gene over a broad range of template concentrations • Everything you need for SYBR Green dye based PCR amplification and detection in a convenient single tube format Power SYBR Green PCR Master Mix contains all of the components excluding the template and primers for superior SYBR Green reagent based real time PCR• The optimized formulation contains highly purified AmpliTaq Gold DNA Polymerase LD to offer greater sensitivity than classic SYBR Green PCR master mix• Power SYBR Green PCR Master Mix contains a blend of dTTP dUTP that is compatible with AmpErase UNG to minimize carryover contamination• Includes a proprietary version of ROX dye an internal passive reference to normalize non PCR related fluorescence fluctuations to minimize well to well variability that result from a variety of causes such as pipetting error and sample evaporation• Power SYBR Green reagent based PCR chemistry easily replaces the existing SYBR Green PCR Master Mix in Applied Biosystems protocols using the same setup and thermal cycling conditions
    Catalog Number:
    4367659
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    Enzymes & Master Mixes for Real-Time PCR|PCR & Real-Time PCR|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Gene Expression Analysis & Genotyping
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    Thermo Fisher power sybr green pcr master mix
    Effect of CIB1 knockdown on de novo KSHV infection. (A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time <t>RT-PCR</t> with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by <t>SYBR</t> green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.
    Alternative Product Try PowerUp SYBR Green Master Mix our newest high performance SYBR dye based master mix for superior performance at a very competitive price With PowerUp SYBR Green Master Mix we ve taken the best of Power SYBR Green PCR Master Mix and added additional capabilities for your gene expression analysis Get superior nucleic acid quantitation with Applied Biosystems Power SYBR Green Master Mix Power SYBR Master Mix offers superior sensitivity and reproducibility detecting as few as two copies of a target gene over a broad range of template concentrations • Everything you need for SYBR Green dye based PCR amplification and detection in a convenient single tube format Power SYBR Green PCR Master Mix contains all of the components excluding the template and primers for superior SYBR Green reagent based real time PCR• The optimized formulation contains highly purified AmpliTaq Gold DNA Polymerase LD to offer greater sensitivity than classic SYBR Green PCR master mix• Power SYBR Green PCR Master Mix contains a blend of dTTP dUTP that is compatible with AmpErase UNG to minimize carryover contamination• Includes a proprietary version of ROX dye an internal passive reference to normalize non PCR related fluorescence fluctuations to minimize well to well variability that result from a variety of causes such as pipetting error and sample evaporation• Power SYBR Green reagent based PCR chemistry easily replaces the existing SYBR Green PCR Master Mix in Applied Biosystems protocols using the same setup and thermal cycling conditions
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    Images

    1) Product Images from "CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells"

    Article Title: CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003941

    Effect of CIB1 knockdown on de novo KSHV infection. (A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by SYBR green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.
    Figure Legend Snippet: Effect of CIB1 knockdown on de novo KSHV infection. (A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by SYBR green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.

    Techniques Used: Infection, shRNA, Isolation, Expressing, Quantitative RT-PCR, Inhibition, Immunofluorescence, Staining, Incubation, SYBR Green Assay, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Over Expression, Western Blot

    2) Product Images from "Increased expression of readthrough acetylcholinesterase variants in the brains of Alzheimer's disease patients"

    Article Title: Increased expression of readthrough acetylcholinesterase variants in the brains of Alzheimer's disease patients

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-160220

    Transcript levels of the “readthrough” acetylcholinesterase (AChE-R) variant are increased in cerebral cortex of AD subjects Relative mRNA levels of the transcripts for AChE-T (or “tailed”), AChE-R (or “readthrought”) and N-AChE (or N-extended) splice variants and for proline-rich membrane anchor 1 (PRiMA-1) were analysed by q RT-PCR in frontal cortex of NDC (n= 22) and AD subjects (n= 19). For AChE transcript analysis specific primers with Power SYBR® Green PCR Master Mix were employed and the specificity of the PCR products was confirmed by dissociation curve analysis. PRiMA-1 transcripts were measured using a specific TaqMan GenExpression Assay with TaqMan PCR Master Mix. Transcript levels were calculated by the comparative 2 −ΔCt method with respect to GAPDH. The results were confirmed in two independent determinations. Mean value ± SEM are represented. *Significantly increased ( p
    Figure Legend Snippet: Transcript levels of the “readthrough” acetylcholinesterase (AChE-R) variant are increased in cerebral cortex of AD subjects Relative mRNA levels of the transcripts for AChE-T (or “tailed”), AChE-R (or “readthrought”) and N-AChE (or N-extended) splice variants and for proline-rich membrane anchor 1 (PRiMA-1) were analysed by q RT-PCR in frontal cortex of NDC (n= 22) and AD subjects (n= 19). For AChE transcript analysis specific primers with Power SYBR® Green PCR Master Mix were employed and the specificity of the PCR products was confirmed by dissociation curve analysis. PRiMA-1 transcripts were measured using a specific TaqMan GenExpression Assay with TaqMan PCR Master Mix. Transcript levels were calculated by the comparative 2 −ΔCt method with respect to GAPDH. The results were confirmed in two independent determinations. Mean value ± SEM are represented. *Significantly increased ( p

    Techniques Used: Variant Assay, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction

    3) Product Images from "Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis"

    Article Title: Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

    Journal: Journal of Virology

    doi: 10.1128/JVI.02119-16

    Hypermutation of the FeLV-B genome and APOBEC3G mRNA expression in human cells. FeLV genomic sequences (segments of Gag and Pol) were cloned by PCR from infected human cells, and individual templates were sequenced and compared to the reference input virus (pFGB clone). APOBEC3G mRNA expression was determined in the same cells (prior to infection) by quantitative real-time PCR (with SYBR green). (A) Representative plots of hypermutation visualized by the online HYPERMUT program ( www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html ), where sequence changes relative to the reference FeLV-B genome are color coded (red, GG→AG; cyan, GA→AA; green, GC→AC; magenta, GT→AT; black, non-G→A). (B) X/Y plots of G→A mutation (per kilobase) against APOBEC3G mRNA levels (where the level in HEK293 cells is taken as 1) with cell lines sorted according to FeLV restriction phenotype. Results for LCLs, which are discordant by virtue of their low levels of infectious virion release ( Table 2 ) but postinfection accumulation of proviral DNA ( Fig. 3 ), are enclosed by a dashed oval. (C) Percentage of G→A mutations that conform to the A3G signature ( 42 ) for all cell lines in which significant levels of mutations were detected. Blue-gray bars, hematopoietic cells; yellow bars, nonhematopoietic cells. (D) Relative levels of APOBEC3G mRNA (on a log 10 scale, with the level in HEK293 cells taken as 1) for all the cell lines tested, sorted into hematopoietic (blue circles) and nonhematopoietic (green circles) cell lines. Nonpermissive cells (nonspreading, with low virion release) are represented by black circles.
    Figure Legend Snippet: Hypermutation of the FeLV-B genome and APOBEC3G mRNA expression in human cells. FeLV genomic sequences (segments of Gag and Pol) were cloned by PCR from infected human cells, and individual templates were sequenced and compared to the reference input virus (pFGB clone). APOBEC3G mRNA expression was determined in the same cells (prior to infection) by quantitative real-time PCR (with SYBR green). (A) Representative plots of hypermutation visualized by the online HYPERMUT program ( www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html ), where sequence changes relative to the reference FeLV-B genome are color coded (red, GG→AG; cyan, GA→AA; green, GC→AC; magenta, GT→AT; black, non-G→A). (B) X/Y plots of G→A mutation (per kilobase) against APOBEC3G mRNA levels (where the level in HEK293 cells is taken as 1) with cell lines sorted according to FeLV restriction phenotype. Results for LCLs, which are discordant by virtue of their low levels of infectious virion release ( Table 2 ) but postinfection accumulation of proviral DNA ( Fig. 3 ), are enclosed by a dashed oval. (C) Percentage of G→A mutations that conform to the A3G signature ( 42 ) for all cell lines in which significant levels of mutations were detected. Blue-gray bars, hematopoietic cells; yellow bars, nonhematopoietic cells. (D) Relative levels of APOBEC3G mRNA (on a log 10 scale, with the level in HEK293 cells taken as 1) for all the cell lines tested, sorted into hematopoietic (blue circles) and nonhematopoietic (green circles) cell lines. Nonpermissive cells (nonspreading, with low virion release) are represented by black circles.

    Techniques Used: Expressing, Genomic Sequencing, Clone Assay, Polymerase Chain Reaction, Infection, Real-time Polymerase Chain Reaction, SYBR Green Assay, Sequencing, Mutagenesis

    4) Product Images from "Cyclophilin A (CypA) Interacts with NF-κB Subunit, p65/RelA, and Contributes to NF-κB Activation Signaling"

    Article Title: Cyclophilin A (CypA) Interacts with NF-κB Subunit, p65/RelA, and Contributes to NF-κB Activation Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096211

    CypA Kd-mediated down-regulation of IL-8, IL-6, and IL-1β expression. IL-8, IL-6, and IL-1β expression levels were determined by a quantitative real-time PCR (qRT-PCR) analysis. One microgram of total RNA was used for cDNA synthesis with Superscript III (Invitrogen) and the oligo(dT)15 primer. qRT-PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) detected by the 7300 Real Time PCR System (Applied Biosystems). The relative expression levels of IL-8, IL-6, and IL-1β mRNA were normalized to the internal reference 18S rRNA.
    Figure Legend Snippet: CypA Kd-mediated down-regulation of IL-8, IL-6, and IL-1β expression. IL-8, IL-6, and IL-1β expression levels were determined by a quantitative real-time PCR (qRT-PCR) analysis. One microgram of total RNA was used for cDNA synthesis with Superscript III (Invitrogen) and the oligo(dT)15 primer. qRT-PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) detected by the 7300 Real Time PCR System (Applied Biosystems). The relative expression levels of IL-8, IL-6, and IL-1β mRNA were normalized to the internal reference 18S rRNA.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay, Polymerase Chain Reaction

    5) Product Images from "MicroRNA modulate alveolar epithelial response to cyclic stretch"

    Article Title: MicroRNA modulate alveolar epithelial response to cyclic stretch

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-154

    Quantitative real-time PCR validation of miRNAs. To validate differential expression of the miRNAs, 4 of the miRNAs with the largest fold-changes (2 up- and 2 down-regulated; see Table 1 ), were validated using quantitative real-time PCR with either Taqman or SYBR Green. In addition, we performed qRT-PCR on miR-423-5p, which was the only miRNA differentially expressed at both 1 and 6 hours, and miR-466f-3p, which was chosen for subsequent investigations. If significant (FDR of 10%), miRNA fold-change on the array is given below the corresponding stretch duration. PCR results are qualitatively consistent in direction and magnitude with the array. N = 4-6/group. ANOVA and post-hoc Tukey results: *p
    Figure Legend Snippet: Quantitative real-time PCR validation of miRNAs. To validate differential expression of the miRNAs, 4 of the miRNAs with the largest fold-changes (2 up- and 2 down-regulated; see Table 1 ), were validated using quantitative real-time PCR with either Taqman or SYBR Green. In addition, we performed qRT-PCR on miR-423-5p, which was the only miRNA differentially expressed at both 1 and 6 hours, and miR-466f-3p, which was chosen for subsequent investigations. If significant (FDR of 10%), miRNA fold-change on the array is given below the corresponding stretch duration. PCR results are qualitatively consistent in direction and magnitude with the array. N = 4-6/group. ANOVA and post-hoc Tukey results: *p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, SYBR Green Assay, Quantitative RT-PCR, Polymerase Chain Reaction

    6) Product Images from "DHMEQ, a novel nuclear factor-?B inhibitor, induces selective depletion of alloreactive or phytohaemagglutinin-stimulated peripheral blood mononuclear cells, decreases production of T helper type 1 cytokines, and blocks maturation of dendritic cells"

    Article Title: DHMEQ, a novel nuclear factor-?B inhibitor, induces selective depletion of alloreactive or phytohaemagglutinin-stimulated peripheral blood mononuclear cells, decreases production of T helper type 1 cytokines, and blocks maturation of dendritic cells

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2007.02755.x

    Effect of dehydroxymethylepoxyquinomicin (DHMEQ) on phytohaemagglutinin (PHA)-stimulated expression of inflammatory cytokine genes in peripheral blood mononuclear cells (PBMC). PBMC form healthy volunteers (a) ( n = 3) or Jurkat cells (b) were pretreated with either DHMEQ (1 μg/ml) or control diluent for 3 hr and then exposed, or not exposed, to PHA (5 μg/ml) for 3 hr. Cells were harvested and RNA was extracted. cDNAs were synthesized and subjected to real-time polyerase chain reaction (PCR) using SYBR Green nucleic acid gel staining solution to measure the levels of interferon (IFN)-γ, interleukin (IL)-2 and tumour necrosis factor (TNF)-α in these cells. Results represent the mean ± standard deviation (SD) of three experiments with triplicate dishes per experimental point. The statistical significance was determined by a one-way analysis of variance (ANOVA) followed by Boneferroni's multiple comparison test.
    Figure Legend Snippet: Effect of dehydroxymethylepoxyquinomicin (DHMEQ) on phytohaemagglutinin (PHA)-stimulated expression of inflammatory cytokine genes in peripheral blood mononuclear cells (PBMC). PBMC form healthy volunteers (a) ( n = 3) or Jurkat cells (b) were pretreated with either DHMEQ (1 μg/ml) or control diluent for 3 hr and then exposed, or not exposed, to PHA (5 μg/ml) for 3 hr. Cells were harvested and RNA was extracted. cDNAs were synthesized and subjected to real-time polyerase chain reaction (PCR) using SYBR Green nucleic acid gel staining solution to measure the levels of interferon (IFN)-γ, interleukin (IL)-2 and tumour necrosis factor (TNF)-α in these cells. Results represent the mean ± standard deviation (SD) of three experiments with triplicate dishes per experimental point. The statistical significance was determined by a one-way analysis of variance (ANOVA) followed by Boneferroni's multiple comparison test.

    Techniques Used: Expressing, Synthesized, Polymerase Chain Reaction, SYBR Green Assay, Staining, Standard Deviation

    7) Product Images from "Differential Expression of Heat Shock Transcription Factors and Heat Shock Proteins after Acute and Chronic Heat Stress in Laying Chickens (Gallus gallus)"

    Article Title: Differential Expression of Heat Shock Transcription Factors and Heat Shock Proteins after Acute and Chronic Heat Stress in Laying Chickens (Gallus gallus)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0102204

    Normalized gene expression of HSF after acute and chronic heat challenges. Expression of heat shock factor (HSF) genes was determined by real time PCR using SYBR green dye and the 2 −ΔΔCt method was used to calculate mRNA level of each gene, where the average mean of the unstressed group was used as the calibrator. Each PCR reaction was conducted in triplicate and the geometric mean of internal references, β-actin and GAPDH, was used to normalize the expression of targets genes. Values are expressed as means ± SE of data from 5–6 individual tissue samples. * indicates significant differences (P
    Figure Legend Snippet: Normalized gene expression of HSF after acute and chronic heat challenges. Expression of heat shock factor (HSF) genes was determined by real time PCR using SYBR green dye and the 2 −ΔΔCt method was used to calculate mRNA level of each gene, where the average mean of the unstressed group was used as the calibrator. Each PCR reaction was conducted in triplicate and the geometric mean of internal references, β-actin and GAPDH, was used to normalize the expression of targets genes. Values are expressed as means ± SE of data from 5–6 individual tissue samples. * indicates significant differences (P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction

    Normalized gene expression of HSP 70 and 90 after acute and chronic heat challenges. Expression of HSP 70 and 90 genes was determined by real time PCR using SYBR green dye and the 2 −ΔΔCt method was used to calculate mRNA level of each gene, where the average mean of the unstressed group was used as the calibrator. Each PCR reaction was conducted in triplicate and the geometric mean of internal references, β-actin and GAPDH, was used to normalize the expression of targets genes. Values are expressed as means ± SE of data from 5–6 individual tissue samples. * indicates significant differences (P
    Figure Legend Snippet: Normalized gene expression of HSP 70 and 90 after acute and chronic heat challenges. Expression of HSP 70 and 90 genes was determined by real time PCR using SYBR green dye and the 2 −ΔΔCt method was used to calculate mRNA level of each gene, where the average mean of the unstressed group was used as the calibrator. Each PCR reaction was conducted in triplicate and the geometric mean of internal references, β-actin and GAPDH, was used to normalize the expression of targets genes. Values are expressed as means ± SE of data from 5–6 individual tissue samples. * indicates significant differences (P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction

    8) Product Images from "Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways"

    Article Title: Regulation of the reserve carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a possible cross-regulation of both signaling pathways

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3832-1

    The influence of pH and Ca 2+ on the crz-1 expression. a Schematic representation of the crz-1 and pac-1 promoters and the respective transcription factors DNA binding sites. The black dots and square boxes indicate the position of PAC-3 and CRZ-1 motifs, respectively. b Expression of crz-1 gene in Δ pac-3 and wild-type strains under normal and alkaline pH ( left graph ) and under different CaCl 2 concentration ( right graph ). Cells from both strains were cultured at pH 5.8 during 24 h and shifted to pH 7.8 during 1 h. Cells from the wild-type strain were cultured at normal pH (0) for 24 h and shifted to 10 or 300 mM CaCl 2 for 30, 60 and 120 min. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers (Additional file 1 : Table S1, qPCR). The tub-2 and act genes were used as the reference genes in the pH and calcium experiments, respectively. At least three biological replicates were run and the data were analyzed in the relative quantification standard curve method. a, b, c: Letters above bars indicate statistical significance; different letters indicate significant differences between two samples and equal letters indicate no significant difference between two samples in the same or different pH (Student’s t -test, P
    Figure Legend Snippet: The influence of pH and Ca 2+ on the crz-1 expression. a Schematic representation of the crz-1 and pac-1 promoters and the respective transcription factors DNA binding sites. The black dots and square boxes indicate the position of PAC-3 and CRZ-1 motifs, respectively. b Expression of crz-1 gene in Δ pac-3 and wild-type strains under normal and alkaline pH ( left graph ) and under different CaCl 2 concentration ( right graph ). Cells from both strains were cultured at pH 5.8 during 24 h and shifted to pH 7.8 during 1 h. Cells from the wild-type strain were cultured at normal pH (0) for 24 h and shifted to 10 or 300 mM CaCl 2 for 30, 60 and 120 min. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers (Additional file 1 : Table S1, qPCR). The tub-2 and act genes were used as the reference genes in the pH and calcium experiments, respectively. At least three biological replicates were run and the data were analyzed in the relative quantification standard curve method. a, b, c: Letters above bars indicate statistical significance; different letters indicate significant differences between two samples and equal letters indicate no significant difference between two samples in the same or different pH (Student’s t -test, P

    Techniques Used: Expressing, Binding Assay, Concentration Assay, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Activated Clotting Time Assay

    The influence of Ca 2+ on the expression levels of some genes related to glycogen and trehalose regulation in the wild-type and Δ pac-3 strains. Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 (0) for 24 h and shifted to 10 mM or 300 mM CaCl 2 for 15, 30 and 60 min. Mycelial samples were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers (Additional file 1 : Table S1, qPCR). The actin gene was used as the reference gene, and the zero sample in the wild-type was used as the reference sample. At least four biological replicates were performed, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. The asterisks indicate significant differences compared to the zero sample in the wild-type or mutant strains (Student’s t-test, P
    Figure Legend Snippet: The influence of Ca 2+ on the expression levels of some genes related to glycogen and trehalose regulation in the wild-type and Δ pac-3 strains. Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 (0) for 24 h and shifted to 10 mM or 300 mM CaCl 2 for 15, 30 and 60 min. Mycelial samples were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers (Additional file 1 : Table S1, qPCR). The actin gene was used as the reference gene, and the zero sample in the wild-type was used as the reference sample. At least four biological replicates were performed, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. The asterisks indicate significant differences compared to the zero sample in the wild-type or mutant strains (Student’s t-test, P

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation, Mutagenesis

    The expression of glycogenic genes in the wild-type and Δ pac-3 mutant strains at normal growth pH (5.8) and alkaline pH (7.8). Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers. The β- tub gene was used as the reference gene, and the wild-type at pH 5.8 was used as the reference sample. At least three biological replicates were performed in triplicate, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c, d: Letters above the bars indicate statistical significance; different letters indicate significant difference between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t-test, P
    Figure Legend Snippet: The expression of glycogenic genes in the wild-type and Δ pac-3 mutant strains at normal growth pH (5.8) and alkaline pH (7.8). Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were collected and used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers. The β- tub gene was used as the reference gene, and the wild-type at pH 5.8 was used as the reference sample. At least three biological replicates were performed in triplicate, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c, d: Letters above the bars indicate statistical significance; different letters indicate significant difference between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t-test, P

    Techniques Used: Expressing, Mutagenesis, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    The expression of trehalose genes in the wild-type and Δ pac-3 strains at normal growth pH (5.8) and alkaline pH (7.8). Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers described in Additional file 1 : Table S1. The β- tub gene was used as the reference gene, and the wild-type pH 5.8 was used as the reference sample. At least four biological replicates were performed in triplicate, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c, d: Letters above the bars indicate statistical significance; different letters indicate significant differences between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t-test, P
    Figure Legend Snippet: The expression of trehalose genes in the wild-type and Δ pac-3 strains at normal growth pH (5.8) and alkaline pH (7.8). Cells from the wild-type and Δ pac-3 strains were cultured at pH 5.8 for 24 h and shifted to pH 7.8 for 1 h. Mycelial samples were used to extract total RNA. Gene expression analysis was performed by RT-qPCR in the StepOnePlus™ Real-Time PCR system (Applied Biosystems) using the Power SYBR® Green and specific primers described in Additional file 1 : Table S1. The β- tub gene was used as the reference gene, and the wild-type pH 5.8 was used as the reference sample. At least four biological replicates were performed in triplicate, and the data were analyzed using the relative quantification standard curve method. Bars indicate the standard deviation from the biological experiments. a, b, c, d: Letters above the bars indicate statistical significance; different letters indicate significant differences between two samples and similar letters indicate no significant difference between two samples at the same or different pH (Student’s t-test, P

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    9) Product Images from "Development and Application of a Universal Hemoplasma Screening Assay Based on the SYBR Green PCR Principle ▿"

    Article Title: Development and Application of a Universal Hemoplasma Screening Assay Based on the SYBR Green PCR Principle ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01478-09

    Melting curve analysis of SYBR green PCR products.
    Figure Legend Snippet: Melting curve analysis of SYBR green PCR products.

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction

    SYBR green PCR assay optimization.
    Figure Legend Snippet: SYBR green PCR assay optimization.

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction

    Melting curve analysis after hemoplasma SYBR green PCR of samples from animals infected singly with a feline, canine, murine, or porcine hemoplasma species. Each curve represents one PCR amplicon and depicts the change in fluorescence during a continuous
    Figure Legend Snippet: Melting curve analysis after hemoplasma SYBR green PCR of samples from animals infected singly with a feline, canine, murine, or porcine hemoplasma species. Each curve represents one PCR amplicon and depicts the change in fluorescence during a continuous

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction, Infection, Amplification, Fluorescence

    Comparison of the universal SYBR green PCR assay with specific TaqMan PCR assays.
    Figure Legend Snippet: Comparison of the universal SYBR green PCR assay with specific TaqMan PCR assays.

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction

    10) Product Images from "Characterisation of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage"

    Article Title: Characterisation of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-015-0689-5

    Real-time quantitative PCR analysis of Ts-clp. After reverse transcription, the cDNA of ML, L6h, L24h, Ad2, Ad3, Ad5, and NBL was used as a template for real-time quantitative PCR using SYBR Green. Each reaction was performed in triplicate on the plate, and the experiment was repeated three times. All the fold changes were relative to the NBL stage. * Data are significantly different from NBL ( P
    Figure Legend Snippet: Real-time quantitative PCR analysis of Ts-clp. After reverse transcription, the cDNA of ML, L6h, L24h, Ad2, Ad3, Ad5, and NBL was used as a template for real-time quantitative PCR using SYBR Green. Each reaction was performed in triplicate on the plate, and the experiment was repeated three times. All the fold changes were relative to the NBL stage. * Data are significantly different from NBL ( P

    Techniques Used: Real-time Polymerase Chain Reaction, SYBR Green Assay

    11) Product Images from "Epigenetic repression of bone morphogenetic protein receptor II expression in scleroderma"

    Article Title: Epigenetic repression of bone morphogenetic protein receptor II expression in scleroderma

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12105

    The effects of BMP2 on anti-apoptotic gene Bcl-xL mRNA. Normal and SSc-MVECs cells were treated with BMP2 (200 ng/ml) for 24 hrs. The mRNA expression levels of Bcl-xL were measured by SYBR Green real-time PCR analysis. The expression level of Bcl-xL was markedly increased in BMP2-treated normal-MVECs, but not in SSc-MVECs. Values are the fold changes compared with expression in the normal control group (ascribed an arbitrary value of 1) and are expressed as mean ± SD from triplicate experiments.* P
    Figure Legend Snippet: The effects of BMP2 on anti-apoptotic gene Bcl-xL mRNA. Normal and SSc-MVECs cells were treated with BMP2 (200 ng/ml) for 24 hrs. The mRNA expression levels of Bcl-xL were measured by SYBR Green real-time PCR analysis. The expression level of Bcl-xL was markedly increased in BMP2-treated normal-MVECs, but not in SSc-MVECs. Values are the fold changes compared with expression in the normal control group (ascribed an arbitrary value of 1) and are expressed as mean ± SD from triplicate experiments.* P

    Techniques Used: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction

    Decreased expression levels of BMPRII in systemic sclerosis (SSc) cells and skin. ( A ) mRNA expression levels of all BMPR family members (BMPR1A, BMPR1B and BMPRII) in normal-MVECs and SSc-MVECs were measured by SYBR Green real-time PCR analysis. Values are the fold increase/decrease compared with expression in the normal control group (ascribed an arbitrary value of 1). There was a significant decrease in BMPRII expression levels in SSc-MVECs * P
    Figure Legend Snippet: Decreased expression levels of BMPRII in systemic sclerosis (SSc) cells and skin. ( A ) mRNA expression levels of all BMPR family members (BMPR1A, BMPR1B and BMPRII) in normal-MVECs and SSc-MVECs were measured by SYBR Green real-time PCR analysis. Values are the fold increase/decrease compared with expression in the normal control group (ascribed an arbitrary value of 1). There was a significant decrease in BMPRII expression levels in SSc-MVECs * P

    Techniques Used: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction

    12) Product Images from "Strand-specific deep sequencing of the transcriptome"

    Article Title: Strand-specific deep sequencing of the transcriptome

    Journal: Genome Research

    doi: 10.1101/gr.094318.109

    Comparative evaluation of deep sequencing. ( A ) Cumulative coverage of read data set, expressed as the ratio of sequenced bases located in annotated genes and the total number of bases in annotated genes. Exhaustive coverage is reached after 14 lanes (Supplemental Fig. 5). ( B ) Comparison of dynamic range of expression measurements (protein coding genes only) on tiling array and DSSS expressed on a log 2 scale. ( C ) Validation of DSSS results using qPCR. DNase-treated RNA was reverse transcribed and subjected to SYBR green real-time PCR. Non-reverse-transcribed controls were included for each gene, as well as a genomic DNA dilution series. DSSS signal corresponds to the log 2 transformed mean number of counts along the gene.
    Figure Legend Snippet: Comparative evaluation of deep sequencing. ( A ) Cumulative coverage of read data set, expressed as the ratio of sequenced bases located in annotated genes and the total number of bases in annotated genes. Exhaustive coverage is reached after 14 lanes (Supplemental Fig. 5). ( B ) Comparison of dynamic range of expression measurements (protein coding genes only) on tiling array and DSSS expressed on a log 2 scale. ( C ) Validation of DSSS results using qPCR. DNase-treated RNA was reverse transcribed and subjected to SYBR green real-time PCR. Non-reverse-transcribed controls were included for each gene, as well as a genomic DNA dilution series. DSSS signal corresponds to the log 2 transformed mean number of counts along the gene.

    Techniques Used: Sequencing, Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Transformation Assay

    13) Product Images from "IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP"

    Article Title: IFI16, a nuclear innate immune DNA sensor, mediates epigenetic silencing of herpesvirus genomes by its association with H3K9 methyltransferases SUV39H1 and GLP

    Journal: eLife

    doi: 10.7554/eLife.49500

    Effect of A366 on KSHV life cycle and the demonstration of IFI16’s association with cellular H3K9 methyltransferase(s) (H3K9 MTase) and recruitment of various H3K9 MTases to the KSHV genome during de novo infection. ( A ) MTT cell viability assay of BCBL-1 cells treated with the H3K9me3 specific chemical inhibitor A366 at different concentrations and different time points. ( B ) q-RT PCR (two-step, sybr Green) of KSHV mRNAs in BCBL-1 cells treated for 72 hr with either vehicle control DMSO or A366 (10 µM and 100 µM). ( C ) WB of different H3 methylations and IFI16 after A366 treatment of BCBL-1 cells. ( D ) H3K9 methyltransferase activity (ng/h/mg) assay. TIME cells were infected with KSHV for 6 or 24 hr followed by isolation of nuclear fraction, benzonase treatment and IP with anti-IFI16 or control IgG in the presence of benzonase using the catch and release method. Elution was performed under non-denaturing conditions to keep the associated H3K9 methyltransferase active. H3K9 methyltransferase activity was assayed in the eluate (Materials and methods). *, p
    Figure Legend Snippet: Effect of A366 on KSHV life cycle and the demonstration of IFI16’s association with cellular H3K9 methyltransferase(s) (H3K9 MTase) and recruitment of various H3K9 MTases to the KSHV genome during de novo infection. ( A ) MTT cell viability assay of BCBL-1 cells treated with the H3K9me3 specific chemical inhibitor A366 at different concentrations and different time points. ( B ) q-RT PCR (two-step, sybr Green) of KSHV mRNAs in BCBL-1 cells treated for 72 hr with either vehicle control DMSO or A366 (10 µM and 100 µM). ( C ) WB of different H3 methylations and IFI16 after A366 treatment of BCBL-1 cells. ( D ) H3K9 methyltransferase activity (ng/h/mg) assay. TIME cells were infected with KSHV for 6 or 24 hr followed by isolation of nuclear fraction, benzonase treatment and IP with anti-IFI16 or control IgG in the presence of benzonase using the catch and release method. Elution was performed under non-denaturing conditions to keep the associated H3K9 methyltransferase active. H3K9 methyltransferase activity was assayed in the eluate (Materials and methods). *, p

    Techniques Used: Infection, MTT Assay, Viability Assay, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Western Blot, Activity Assay, Isolation

    14) Product Images from "FOXK1 interaction with FHL2 promotes proliferation, invasion and metastasis in colorectal cancer"

    Article Title: FOXK1 interaction with FHL2 promotes proliferation, invasion and metastasis in colorectal cancer

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2016.68

    FOXK1 expression is higher in human CRC Tissues. ( a ) Expression pattern of FOXK1 mRNA in normal and tumour tissues. FOXK1 mRNA expression in various types of cancer was searched in the GENT database (available at http://medical-genomics.kribb.re.kr/GENT/ ). Boxes represent the median and the 25th and 75th percentiles; dots represent outliers. Red boxes represent tumour tissues; green boxes represent normal tissues. Red and green dashed lines represent the average value of all tumour and normal tissues, respectively. The asterisk indicates the significant increase of FOXK1 expression in colon tumours compared with normal tissues. FOXK1 mRNA expression of colon tissue: blue dotted lines. ( b ) Expression of FOXK1 by qRT–PCR in 10 pairs of colon cancer (tumour) and matched non-cancerous colonic tissues (normal). All of these experiments were repeated three times with identical findings. ( c ) On average, higher expression level of FOXK1 was found in tumour than in normal tissues ( n =10). **** P
    Figure Legend Snippet: FOXK1 expression is higher in human CRC Tissues. ( a ) Expression pattern of FOXK1 mRNA in normal and tumour tissues. FOXK1 mRNA expression in various types of cancer was searched in the GENT database (available at http://medical-genomics.kribb.re.kr/GENT/ ). Boxes represent the median and the 25th and 75th percentiles; dots represent outliers. Red boxes represent tumour tissues; green boxes represent normal tissues. Red and green dashed lines represent the average value of all tumour and normal tissues, respectively. The asterisk indicates the significant increase of FOXK1 expression in colon tumours compared with normal tissues. FOXK1 mRNA expression of colon tissue: blue dotted lines. ( b ) Expression of FOXK1 by qRT–PCR in 10 pairs of colon cancer (tumour) and matched non-cancerous colonic tissues (normal). All of these experiments were repeated three times with identical findings. ( c ) On average, higher expression level of FOXK1 was found in tumour than in normal tissues ( n =10). **** P

    Techniques Used: Expressing, Quantitative RT-PCR

    15) Product Images from "Fc?R1-Mediated Mast Cell Reactivity Is Amplified through Prolonged Toll-Like Receptor-Ligand Treatment"

    Article Title: Fc?R1-Mediated Mast Cell Reactivity Is Amplified through Prolonged Toll-Like Receptor-Ligand Treatment

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043547

    Relative expression of TLRs (1–9) in CTLMC and MLMC. RNA purified from CTLMC and MLMC was subjected to quantitative real-time PCR using SYBR Green for the indicated TLRs. The relative gene expression levels were determined in triplicates by difference in Ct numbers and results were described as in relation to Gmol β actin. Data are plotted as mean ± SEM and derived from 4–6 independent experiments.
    Figure Legend Snippet: Relative expression of TLRs (1–9) in CTLMC and MLMC. RNA purified from CTLMC and MLMC was subjected to quantitative real-time PCR using SYBR Green for the indicated TLRs. The relative gene expression levels were determined in triplicates by difference in Ct numbers and results were described as in relation to Gmol β actin. Data are plotted as mean ± SEM and derived from 4–6 independent experiments.

    Techniques Used: Expressing, Purification, Real-time Polymerase Chain Reaction, SYBR Green Assay, Derivative Assay

    16) Product Images from "TRAIL DR5-CTSB crosstalk participates in breast cancer autophagy initiated by SAHA"

    Article Title: TRAIL DR5-CTSB crosstalk participates in breast cancer autophagy initiated by SAHA

    Journal: Cell Death Discovery

    doi: 10.1038/cddiscovery.2017.52

    The effects of SAHA and TRAIL DR5 on the expressions of autophagy-related genes in cancer cells. RNA was isolated from MDA-MB-231 or MCF-7 cells using high pure RNA isolation kit following manufacturer’s instructions. To obtain the first-strand cDNA, transcriptor first strand cDNA synthesis kit was used and cDNA was as a template in real-time PCR reactions with power SYBR green PCR master mix. Exprofile human autophagy Gene qPCR array kit was employed to describe related mRNA expression. Relative changes of gene expression in the array were calculated using the 2 −ΔΔCt (threshold cycle) method. ( a – c ). MDA-MB-231 cells, ( d – f ). MCF-7 cells, ( g ). Related data analysis. Relative quantitative values represent mean±S.E.M.; * represents statistical significance of P
    Figure Legend Snippet: The effects of SAHA and TRAIL DR5 on the expressions of autophagy-related genes in cancer cells. RNA was isolated from MDA-MB-231 or MCF-7 cells using high pure RNA isolation kit following manufacturer’s instructions. To obtain the first-strand cDNA, transcriptor first strand cDNA synthesis kit was used and cDNA was as a template in real-time PCR reactions with power SYBR green PCR master mix. Exprofile human autophagy Gene qPCR array kit was employed to describe related mRNA expression. Relative changes of gene expression in the array were calculated using the 2 −ΔΔCt (threshold cycle) method. ( a – c ). MDA-MB-231 cells, ( d – f ). MCF-7 cells, ( g ). Related data analysis. Relative quantitative values represent mean±S.E.M.; * represents statistical significance of P

    Techniques Used: Isolation, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction, Expressing

    17) Product Images from "Perivascular Stem Cells Suppress Inflammasome Activation during Inflammatory Responses in Macrophages"

    Article Title: Perivascular Stem Cells Suppress Inflammasome Activation during Inflammatory Responses in Macrophages

    Journal: International Journal of Stem Cells

    doi: 10.15283/ijsc19115

    PVC-CM reduces inflammation and ER stress in in macrophages of MSU-induced peritonitis. (A) Schematic diagram of experiments using the peritonitis mouse model (n=8 per group). (B) PECs were determined by counting exudate cells with a cell counter. (C) Cytospins of isolated PECs were stained with Wright-Giemsa stain. (D, E) Relative mRNA levels of inflammatory (IL-1 α , IL-1 β , NLRP3, and Casp1) and ER stress-related genes (ATF6, PERK, elk2 α , and IRE-1) were analyzed using SYBR green-based quantitative real-time PCR. Error bars indicate SD (*p
    Figure Legend Snippet: PVC-CM reduces inflammation and ER stress in in macrophages of MSU-induced peritonitis. (A) Schematic diagram of experiments using the peritonitis mouse model (n=8 per group). (B) PECs were determined by counting exudate cells with a cell counter. (C) Cytospins of isolated PECs were stained with Wright-Giemsa stain. (D, E) Relative mRNA levels of inflammatory (IL-1 α , IL-1 β , NLRP3, and Casp1) and ER stress-related genes (ATF6, PERK, elk2 α , and IRE-1) were analyzed using SYBR green-based quantitative real-time PCR. Error bars indicate SD (*p

    Techniques Used: Isolation, Staining, Giemsa Stain, SYBR Green Assay, Real-time Polymerase Chain Reaction

    18) Product Images from "Thymic PTH Increases After Thyroparathyroidectomy in C57BL/KaLwRij Mice"

    Article Title: Thymic PTH Increases After Thyroparathyroidectomy in C57BL/KaLwRij Mice

    Journal: Endocrinology

    doi: 10.1210/en.2017-03083

    The thymus produces a significant number of Pth transcripts that increase after TPTX. (A) The thymus produced Pth transcripts. Mice in which serum PTH levels recovered (n = 10) were euthanized, and the heart, lung, liver, spleen, kidney, pancreas, testis or ovary, hypothalamus, pituitary gland, thymus, and adrenal glands were harvested. RNA qRT-PCR was performed on the tissue samples using SYBR Green/Taqman Master Mix with custom primers for the mouse Pth transcript and a commercial probe-set for 18S rRNA (endogenous control). Expression data were normalized to 18S rRNA, and fold changes of expression against arbitrary numbers were calculated using the 2 − ΔΔ Ct ). (B) Pth transcripts increased after TPTX. Thymi from unoperated mice (control) were harvested, and the same procedures were performed as in (A). qRT-PCR was performed as described with a commercial probe (gray) and custom probes (black). Student t test was performed. ** P
    Figure Legend Snippet: The thymus produces a significant number of Pth transcripts that increase after TPTX. (A) The thymus produced Pth transcripts. Mice in which serum PTH levels recovered (n = 10) were euthanized, and the heart, lung, liver, spleen, kidney, pancreas, testis or ovary, hypothalamus, pituitary gland, thymus, and adrenal glands were harvested. RNA qRT-PCR was performed on the tissue samples using SYBR Green/Taqman Master Mix with custom primers for the mouse Pth transcript and a commercial probe-set for 18S rRNA (endogenous control). Expression data were normalized to 18S rRNA, and fold changes of expression against arbitrary numbers were calculated using the 2 − ΔΔ Ct ). (B) Pth transcripts increased after TPTX. Thymi from unoperated mice (control) were harvested, and the same procedures were performed as in (A). qRT-PCR was performed as described with a commercial probe (gray) and custom probes (black). Student t test was performed. ** P

    Techniques Used: Produced, Mouse Assay, Quantitative RT-PCR, SYBR Green Assay, Expressing

    19) Product Images from "Amyloid precursor protein glycosylation is altered in the brain of patients with Alzheimer’s disease"

    Article Title: Amyloid precursor protein glycosylation is altered in the brain of patients with Alzheimer’s disease

    Journal: Alzheimer's Research & Therapy

    doi: 10.1186/s13195-020-00664-9

    Increased APP mRNA in the frontal cortex of AD patients. Relative mRNA expression of transcripts for a total APP, and the b APP-KPI and APP-695 splice variants, analyzed by q RT-PCR in frontal cortex tissue from NDC ( n = 7) and AD subjects (Braak stages V–VI, – = 7). To analyze APP transcripts, specific Power SYBR® Green PCR Master Mix primers were employed and the specificity of the PCR products was confirmed by analyzing the dissociation curves. Transcript levels were calculated by the comparative 2 −ΔCt method with respect to 18S rRNA from the same cDNA and expressed as the mean ± SEM: p
    Figure Legend Snippet: Increased APP mRNA in the frontal cortex of AD patients. Relative mRNA expression of transcripts for a total APP, and the b APP-KPI and APP-695 splice variants, analyzed by q RT-PCR in frontal cortex tissue from NDC ( n = 7) and AD subjects (Braak stages V–VI, – = 7). To analyze APP transcripts, specific Power SYBR® Green PCR Master Mix primers were employed and the specificity of the PCR products was confirmed by analyzing the dissociation curves. Transcript levels were calculated by the comparative 2 −ΔCt method with respect to 18S rRNA from the same cDNA and expressed as the mean ± SEM: p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Polymerase Chain Reaction

    20) Product Images from "Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR"

    Article Title: Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/s12276-020-0452-7

    Improvement of the real-time PCR detection protocol for SARS-CoV-2. Real-time PCR was performed using the SARS-CoV-2 primer sets SARS-CoV-2_IBS_E2, SARS-CoV-2_IBS_RdRP2, SARS-CoV-2_IBS_S2, and SARS-CoV-2_IBS_N1. The GAPDH primer set was used as the IPC primer set. Each row represents each primer set. On the left, each graph represents an amplification plot, which shows the variation of log (ΔRn) values against the PCR cycle number. The Y axis represents the normalized reporter value (Rn), which was calculated as the fluorescence signal from SYBR Green normalized to the fluorescence signal of a reference dye. The graphs on the right represent the melting curve plots, which display data collected during a melting curve stage. Peaks in the melting curve may indicate the melting temperature ( T m ) of a target or identify nonspecific PCR amplification. On the Y axis, the derivative reporter (−Rn′) was calculated as the negative first derivative of Rn generated by the reporter during PCR amplification. The green curve is for the Volunteer U cDNA sample. The blue curve is for the SARS-CoV-2 cDNA. The purple curve is for the no-template condition. All data are represented as the mean ± S.E.M.
    Figure Legend Snippet: Improvement of the real-time PCR detection protocol for SARS-CoV-2. Real-time PCR was performed using the SARS-CoV-2 primer sets SARS-CoV-2_IBS_E2, SARS-CoV-2_IBS_RdRP2, SARS-CoV-2_IBS_S2, and SARS-CoV-2_IBS_N1. The GAPDH primer set was used as the IPC primer set. Each row represents each primer set. On the left, each graph represents an amplification plot, which shows the variation of log (ΔRn) values against the PCR cycle number. The Y axis represents the normalized reporter value (Rn), which was calculated as the fluorescence signal from SYBR Green normalized to the fluorescence signal of a reference dye. The graphs on the right represent the melting curve plots, which display data collected during a melting curve stage. Peaks in the melting curve may indicate the melting temperature ( T m ) of a target or identify nonspecific PCR amplification. On the Y axis, the derivative reporter (−Rn′) was calculated as the negative first derivative of Rn generated by the reporter during PCR amplification. The green curve is for the Volunteer U cDNA sample. The blue curve is for the SARS-CoV-2 cDNA. The purple curve is for the no-template condition. All data are represented as the mean ± S.E.M.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Fluorescence, SYBR Green Assay, Generated

    21) Product Images from "Rotavirus and Serotonin Cross-Talk in Diarrhoea"

    Article Title: Rotavirus and Serotonin Cross-Talk in Diarrhoea

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0159660

    Rotavirus down-regulates SERT mRNA in ileum of infected mice. The small intestines of infected and uninfected mice were collected 24 and 48 h p.i and SERT and GAPDH mRNA levels quantified at each time-point by SYBR Green based real-time PCR. A significant down-regulation of SERT mRNA was found at 48 h p.i in ileum of infected compared to uninfected mice. Relative fold- change was set in relation to uninfected pups. Statistical analyses were done using Kruskal-Wallis and Bonferroni’s multiple comparison test. Data is presented as means + SEM. * = p
    Figure Legend Snippet: Rotavirus down-regulates SERT mRNA in ileum of infected mice. The small intestines of infected and uninfected mice were collected 24 and 48 h p.i and SERT and GAPDH mRNA levels quantified at each time-point by SYBR Green based real-time PCR. A significant down-regulation of SERT mRNA was found at 48 h p.i in ileum of infected compared to uninfected mice. Relative fold- change was set in relation to uninfected pups. Statistical analyses were done using Kruskal-Wallis and Bonferroni’s multiple comparison test. Data is presented as means + SEM. * = p

    Techniques Used: Infection, Mouse Assay, SYBR Green Assay, Real-time Polymerase Chain Reaction

    22) Product Images from "Pan-human coronavirus and human bocavirus SYBR Green and TaqMan PCR assays; use in studying influenza A viruses co-infection and risk of hospitalization"

    Article Title: Pan-human coronavirus and human bocavirus SYBR Green and TaqMan PCR assays; use in studying influenza A viruses co-infection and risk of hospitalization

    Journal: Infection

    doi: 10.1007/s15010-014-0710-5

    Polyacrylamide gel electrophoresis of hCoV PCR products. PCR products from two experiments, one where each well contained 3 μl of hCoV229E RNA (Exp1) and another 5 μl, to increase yield (Exp2) and products from RNA produced from the pEX-A vector. HypperLadder V marks are from bottom, 25, 50, 75, 100 bp up to 500. The marks at 50 bp are due to primer dimmers which are known to form in SYBR Green PCRs
    Figure Legend Snippet: Polyacrylamide gel electrophoresis of hCoV PCR products. PCR products from two experiments, one where each well contained 3 μl of hCoV229E RNA (Exp1) and another 5 μl, to increase yield (Exp2) and products from RNA produced from the pEX-A vector. HypperLadder V marks are from bottom, 25, 50, 75, 100 bp up to 500. The marks at 50 bp are due to primer dimmers which are known to form in SYBR Green PCRs

    Techniques Used: Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Produced, Plasmid Preparation, SYBR Green Assay

    23) Product Images from "Nbn and Atm Cooperate in a Tissue and Developmental Stage-Specific Manner to Prevent Double Strand Breaks and Apoptosis in Developing Brain and Eye"

    Article Title: Nbn and Atm Cooperate in a Tissue and Developmental Stage-Specific Manner to Prevent Double Strand Breaks and Apoptosis in Developing Brain and Eye

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069209

    Nbn-deficiency leads to Atm-dependent apoptotic cell death in developing retina, but cell fate specification and differentiation are normal in the Nbn/Atm-deficient retina. ( A ) Relative expression of Nbn mRNA at different stages of mouse retinal development was analyzed by real time RT-PCR using SYBR green. No significant difference in the levels of Nbn mRNA expression was detected within the stages analyzed. Gene expression was normalized to Gapdh mRNA ( Gapdh ). ( B ) Quantification of γ-H2AX positive cells within retina at E15.5 ( Nbn Ctrl , n = 3; Nbn Nes-Cre , n = 4; and Nbn/Atm Nes-Cre , n = 3) and E17.5 ( Nbn Ctrl , n = 2; Nbn Nes-Cre , n = 3; and Nbn/Atm Nes-Cre , n = 3). ( C ) Quantification of H3-P positive cells per mm 2 of retinal tissue at E15. 5 ( Nbn Ctrl , n = 8; Nbn Nes-Cre , n = 6 and Nbn/Atm Nes-Cre , n = 5) and at E17.5 ( Nbn Ctrl , n = 13; Nbn Nes-Cre , n = 3 and Nbn/Atm Nes-Cre , n = 6). ( D ) Quantification of BrdU positive cells within retina at E15.5 ( Nbn Ctrl , n = 10; Nbn Nes-Cre , n = 6; and Nbn/Atm Nes-Cre , n = 12) and E17.5 ( Nbn Ctrl , n = 8; Nbn Nes-Cre , n = 3; and Nbn/Atm Nes-Cre , n = 2). Error bars indicate SEM (* p
    Figure Legend Snippet: Nbn-deficiency leads to Atm-dependent apoptotic cell death in developing retina, but cell fate specification and differentiation are normal in the Nbn/Atm-deficient retina. ( A ) Relative expression of Nbn mRNA at different stages of mouse retinal development was analyzed by real time RT-PCR using SYBR green. No significant difference in the levels of Nbn mRNA expression was detected within the stages analyzed. Gene expression was normalized to Gapdh mRNA ( Gapdh ). ( B ) Quantification of γ-H2AX positive cells within retina at E15.5 ( Nbn Ctrl , n = 3; Nbn Nes-Cre , n = 4; and Nbn/Atm Nes-Cre , n = 3) and E17.5 ( Nbn Ctrl , n = 2; Nbn Nes-Cre , n = 3; and Nbn/Atm Nes-Cre , n = 3). ( C ) Quantification of H3-P positive cells per mm 2 of retinal tissue at E15. 5 ( Nbn Ctrl , n = 8; Nbn Nes-Cre , n = 6 and Nbn/Atm Nes-Cre , n = 5) and at E17.5 ( Nbn Ctrl , n = 13; Nbn Nes-Cre , n = 3 and Nbn/Atm Nes-Cre , n = 6). ( D ) Quantification of BrdU positive cells within retina at E15.5 ( Nbn Ctrl , n = 10; Nbn Nes-Cre , n = 6; and Nbn/Atm Nes-Cre , n = 12) and E17.5 ( Nbn Ctrl , n = 8; Nbn Nes-Cre , n = 3; and Nbn/Atm Nes-Cre , n = 2). Error bars indicate SEM (* p

    Techniques Used: Expressing, Quantitative RT-PCR, SYBR Green Assay

    24) Product Images from "The Porphyromonas gingivalis Ferric Uptake Regulator Orthologue Binds Hemin and Regulates Hemin-Responsive Biofilm Development"

    Article Title: The Porphyromonas gingivalis Ferric Uptake Regulator Orthologue Binds Hemin and Regulates Hemin-Responsive Biofilm Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111168

    EMSA of Zn(II)Har150 binding to DNA. (A). Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Lanes 2–7, 9 11 all contain 500 ng (0.2 µM) of a 240 bp PCR product encompassing the 33277 dnaA promoter sequence (PGN_0001). Additionally, lane 3 has 1 µg (2.8 µM) Zn(II)Har150; lane 4, 2 µg (5.6 µM) Zn(II)Har150; lane 5, 3 µg (8.4 µM) Zn(II)Har150; lane 6, 4 µg (11.2 µM) Zn(II)Har150 and lane 7, 5 µg (14 µM) Zn(II)Har150. Lane 2 contains DNA only whereas Lane 8 contains 5 µg (14 µM) Zn(II)Har150 only. Lanes 9 – 12 contain the negative control protein for DNA binding, BSA, where there is 5 µg BSA in lanes 9 10, and 3 µg BSA in lanes 11 12. (B). Agarose gel electrophoresis stained with SimplyBlue SafeStain (Life Technologies) for visualizing protein following DNA visualisation. Lanes are as described in (A). The position of the anode (+) and cathode (-) are noted. (C). EMSA competition experiment where an excess of unlabeled dnaA promoter DNA (1250 ng) competed with 250 ng (0.1 µM) FAM-labeled dnaA promoter DNA for binding to 3 µg (8.4 µM) Zn(II)Har150 (lane 3). Lane 1 contains 250 ng FAM-labeled DNA only, whereas lane 2 contains 250 ng FAM-labeled DNA bound to 3 µg Zn(II)Har150. Visualised is the fluorescence of the FAM-labeled DNA after agarose gel electrophoresis (D) EMSA experiment where the promoter-containing DNA of PGN_1308 (lanes 1–3) was shifted by its cognate transcriptional repressor (lane 2) but not by Zn(II)Har150 (lane 3). (E). Inhibition of Zn(II)Har150 DNA binding by hemin. The addition of increasing concentrations of hemin (lane 3, 0 µM; lane 4, 14 µM; lane 5, 70 µM; lane 6, 140 µM) to a constant amount of DNA (500 ng, lanes 2–6) and Zn(II)Har150 (14 µM, lanes 3–6) resulted in increasing inhibition of DNA binding by Zn(II)Har150. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA.
    Figure Legend Snippet: EMSA of Zn(II)Har150 binding to DNA. (A). Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Lanes 2–7, 9 11 all contain 500 ng (0.2 µM) of a 240 bp PCR product encompassing the 33277 dnaA promoter sequence (PGN_0001). Additionally, lane 3 has 1 µg (2.8 µM) Zn(II)Har150; lane 4, 2 µg (5.6 µM) Zn(II)Har150; lane 5, 3 µg (8.4 µM) Zn(II)Har150; lane 6, 4 µg (11.2 µM) Zn(II)Har150 and lane 7, 5 µg (14 µM) Zn(II)Har150. Lane 2 contains DNA only whereas Lane 8 contains 5 µg (14 µM) Zn(II)Har150 only. Lanes 9 – 12 contain the negative control protein for DNA binding, BSA, where there is 5 µg BSA in lanes 9 10, and 3 µg BSA in lanes 11 12. (B). Agarose gel electrophoresis stained with SimplyBlue SafeStain (Life Technologies) for visualizing protein following DNA visualisation. Lanes are as described in (A). The position of the anode (+) and cathode (-) are noted. (C). EMSA competition experiment where an excess of unlabeled dnaA promoter DNA (1250 ng) competed with 250 ng (0.1 µM) FAM-labeled dnaA promoter DNA for binding to 3 µg (8.4 µM) Zn(II)Har150 (lane 3). Lane 1 contains 250 ng FAM-labeled DNA only, whereas lane 2 contains 250 ng FAM-labeled DNA bound to 3 µg Zn(II)Har150. Visualised is the fluorescence of the FAM-labeled DNA after agarose gel electrophoresis (D) EMSA experiment where the promoter-containing DNA of PGN_1308 (lanes 1–3) was shifted by its cognate transcriptional repressor (lane 2) but not by Zn(II)Har150 (lane 3). (E). Inhibition of Zn(II)Har150 DNA binding by hemin. The addition of increasing concentrations of hemin (lane 3, 0 µM; lane 4, 14 µM; lane 5, 70 µM; lane 6, 140 µM) to a constant amount of DNA (500 ng, lanes 2–6) and Zn(II)Har150 (14 µM, lanes 3–6) resulted in increasing inhibition of DNA binding by Zn(II)Har150. Lane 1, HyperladderI (Bioline) DNA size markers in bp. Agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Life Technologies) for visualizing DNA.

    Techniques Used: Binding Assay, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction, Sequencing, Negative Control, Labeling, Fluorescence, Inhibition

    25) Product Images from "A transcriptome-based protein network that identifies new therapeutic targets in colorectal cancer"

    Article Title: A transcriptome-based protein network that identifies new therapeutic targets in colorectal cancer

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4139-y

    Network based on a restricted number of genes after selection of topological parameters. a Network based on 57 genes showing a node degree superior to 11 (median of node degree of 111 genes-based network). b Network based on 32 genes showing a node degree superior to 11 and a clustering coefficient superior to 0.55 (median of clustering coefficient of 111 genes-based network). Blue color of edges indicated a physical interaction between 2 nodes. The thickness of edges was correlated with the score confidence (large for a high score). The node shape illustrated the specific PCR array: Apoptosis: square, Cancer Pathway: hexagon, Lipoprotein signaling and cholesterol metabolism: circles, Drug metabolism: diamond and Wnt pathway: octagon. The color intensity of nodes was related to transcriptome deregulation (red for up-regulation and green for down-regulation in CRC as compared to NT). Genes showing a deregulation in more than 75% of CRC were characterized by bold line of shape node
    Figure Legend Snippet: Network based on a restricted number of genes after selection of topological parameters. a Network based on 57 genes showing a node degree superior to 11 (median of node degree of 111 genes-based network). b Network based on 32 genes showing a node degree superior to 11 and a clustering coefficient superior to 0.55 (median of clustering coefficient of 111 genes-based network). Blue color of edges indicated a physical interaction between 2 nodes. The thickness of edges was correlated with the score confidence (large for a high score). The node shape illustrated the specific PCR array: Apoptosis: square, Cancer Pathway: hexagon, Lipoprotein signaling and cholesterol metabolism: circles, Drug metabolism: diamond and Wnt pathway: octagon. The color intensity of nodes was related to transcriptome deregulation (red for up-regulation and green for down-regulation in CRC as compared to NT). Genes showing a deregulation in more than 75% of CRC were characterized by bold line of shape node

    Techniques Used: Selection, Polymerase Chain Reaction

    Network building from deregulated genes in different types of PCR arrays. Interactions between sets of deregulated genes identified by transcriptome comparison of CRC and NT were retrieved from the STRING database (version 10.5) for 5 different PCR arrays: a ) Apoptosis, b ) Cancer pathway, c ) Lipoprotein signaling and cholesterol metabolism, d ) Drug metabolism and e ) Wnt pathway. The interactions include direct (physical) and indirect (functional) associations. All interaction sources questioned by STRING were used and the minimum required interaction score was 0.4 (medium confidence). The blue color of edges indicated a physical interaction between 2 nodes. The thickness of edges was correlated with score confidence (large for high score). Color intensity of nodes was related to transcriptome deregulation (red for up-regulation and green for down-regulation in CRC as compared to NT). Genes showing a deregulation in more than 75% of CRC were characterized by a bold line of shape node. Star symbols indicated the node showing the highest number of interactions in the network
    Figure Legend Snippet: Network building from deregulated genes in different types of PCR arrays. Interactions between sets of deregulated genes identified by transcriptome comparison of CRC and NT were retrieved from the STRING database (version 10.5) for 5 different PCR arrays: a ) Apoptosis, b ) Cancer pathway, c ) Lipoprotein signaling and cholesterol metabolism, d ) Drug metabolism and e ) Wnt pathway. The interactions include direct (physical) and indirect (functional) associations. All interaction sources questioned by STRING were used and the minimum required interaction score was 0.4 (medium confidence). The blue color of edges indicated a physical interaction between 2 nodes. The thickness of edges was correlated with score confidence (large for high score). Color intensity of nodes was related to transcriptome deregulation (red for up-regulation and green for down-regulation in CRC as compared to NT). Genes showing a deregulation in more than 75% of CRC were characterized by a bold line of shape node. Star symbols indicated the node showing the highest number of interactions in the network

    Techniques Used: Polymerase Chain Reaction, Functional Assay

    26) Product Images from "Developmental attenuation of N-methyl-D-aspartate receptor subunit expression by microRNAs"

    Article Title: Developmental attenuation of N-methyl-D-aspartate receptor subunit expression by microRNAs

    Journal: Neural Development

    doi: 10.1186/s13064-015-0047-5

    a , b . Relative mRNA levels obtained for Grin2a and Grin2b in rat hippocampal cultures . RNA was extracted between DIV3 (Day In Vitro 3) and DIV20. The values represented are normalized to GAPDH by SYBR qRT-PCR. The expression of their corresponding miRNAs (miR-19a and miR-539) are also represented and normalized to U6. c . Protein expression obtained for GluN2A, GluN2B and the loading control β-actin by Western-Blot. d . Expression of the GluN2A and GluN2B proteins after miRNA inhibitor treatment by Western blot. At DIV8, the rat hippocampal neurons were treated with 25 nM, 50 nM or 75 nM of miR-19a, miR-539 inhibitors, with a control (Ctrl) or untreated (Un.).Data in histograms were quantified with Image J software (n = 3). The values for the protein levels are normalized to β-actin
    Figure Legend Snippet: a , b . Relative mRNA levels obtained for Grin2a and Grin2b in rat hippocampal cultures . RNA was extracted between DIV3 (Day In Vitro 3) and DIV20. The values represented are normalized to GAPDH by SYBR qRT-PCR. The expression of their corresponding miRNAs (miR-19a and miR-539) are also represented and normalized to U6. c . Protein expression obtained for GluN2A, GluN2B and the loading control β-actin by Western-Blot. d . Expression of the GluN2A and GluN2B proteins after miRNA inhibitor treatment by Western blot. At DIV8, the rat hippocampal neurons were treated with 25 nM, 50 nM or 75 nM of miR-19a, miR-539 inhibitors, with a control (Ctrl) or untreated (Un.).Data in histograms were quantified with Image J software (n = 3). The values for the protein levels are normalized to β-actin

    Techniques Used: In Vitro, Quantitative RT-PCR, Expressing, Western Blot, Software

    27) Product Images from "Down-regulation of kallikrein-related peptidase 5 (KLK5) expression in breast cancer patients: a biomarker for the differential diagnosis of breast lesions"

    Article Title: Down-regulation of kallikrein-related peptidase 5 (KLK5) expression in breast cancer patients: a biomarker for the differential diagnosis of breast lesions

    Journal: Clinical proteomics

    doi: 10.1186/1559-0275-8-5

    Relative quantification of KLK5 expression in breast specimens by SYBR Green fluorescence-based Real-Time PCR . Separate calibration curves for KLK5 and HPRT1 expression were constructed from serial dilutions of BT20 breast cancer cells' total cDNA.
    Figure Legend Snippet: Relative quantification of KLK5 expression in breast specimens by SYBR Green fluorescence-based Real-Time PCR . Separate calibration curves for KLK5 and HPRT1 expression were constructed from serial dilutions of BT20 breast cancer cells' total cDNA.

    Techniques Used: Expressing, SYBR Green Assay, Fluorescence, Real-time Polymerase Chain Reaction, Construct

    28) Product Images from "Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles"

    Article Title: Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles

    Journal: BMC Biology

    doi: 10.1186/s12915-018-0529-0

    a Copy number data from quantitative SYBR Green PCR assays for Abat , Kctd7 , Slc2a12 , and Uqcr10 , using primers following the design introduced in Fig. 2d for both 5′ and 3′ loxP ssODN donors. All mice screened for each conditional null attempt were heterozygous conditional null mice from the N1 generation that were sequence confirmed for the conditional allele. Negative control samples were CRISPR-targeted N1 mice that were wild-type for the allele being analyzed. b, c Copy number data from TaqMan ® Copy Number assays for ( b ) Slc2a12 and Smc1a (paired ssODN conditional null targeting attempts) and ( c ) Eif2s2 and Cd44 (single lssDNA conditional null targeting attempts) using N1 progeny from a single founder. Genotypes are listed with the mouse ID. Animals with an asterisk to the right of the genotype were sequence-confirmed for the conditional null allele. Of note, Smc1a is located on the X chromosome, thereby males only have one copy of the gene. As in ( a ), negative control samples were CRISPR-targeted N1 mice that were wild-type for the allele being analyzed
    Figure Legend Snippet: a Copy number data from quantitative SYBR Green PCR assays for Abat , Kctd7 , Slc2a12 , and Uqcr10 , using primers following the design introduced in Fig. 2d for both 5′ and 3′ loxP ssODN donors. All mice screened for each conditional null attempt were heterozygous conditional null mice from the N1 generation that were sequence confirmed for the conditional allele. Negative control samples were CRISPR-targeted N1 mice that were wild-type for the allele being analyzed. b, c Copy number data from TaqMan ® Copy Number assays for ( b ) Slc2a12 and Smc1a (paired ssODN conditional null targeting attempts) and ( c ) Eif2s2 and Cd44 (single lssDNA conditional null targeting attempts) using N1 progeny from a single founder. Genotypes are listed with the mouse ID. Animals with an asterisk to the right of the genotype were sequence-confirmed for the conditional null allele. Of note, Smc1a is located on the X chromosome, thereby males only have one copy of the gene. As in ( a ), negative control samples were CRISPR-targeted N1 mice that were wild-type for the allele being analyzed

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction, Mouse Assay, Sequencing, Negative Control, CRISPR

    Screening strategies for HDR and NHEJ alleles, and random ODN insertion. Relative positions of the primers and approximate sizes of PCR products are listed below each allele. Scissors represent target sites. a Genotyping schemes for detecting loxP donor sequences. Orange triangles represent loxP sites, with representative homology sequence color coded on blue DNA strand. b Genotyping for NHEJ events utilizes a three-primer system, with P1 being shared between P2 and P3. Primers P1 and P3 reside between 100 to 200 bp outside of the target site (an average deletion product size is depicted). The P1 + P3 primer pair may not always amplify a wild-type product, if the target sequences are too far apart. 1400 bp represents the average distance between loxP insertion sites. c Random ODN insertion PCR primers reside internal to homology arm sequence, and will amplify the expected size product if the ssODN donor has been incorporated elsewhere in the genome, away from the critical exon, in addition to the on-target locus. d Primers for the homology arm screen were used in a SYBR-green quantitative PCR reaction from DNA samples from the N1 generation, using β-actin as a two-copy normalization control
    Figure Legend Snippet: Screening strategies for HDR and NHEJ alleles, and random ODN insertion. Relative positions of the primers and approximate sizes of PCR products are listed below each allele. Scissors represent target sites. a Genotyping schemes for detecting loxP donor sequences. Orange triangles represent loxP sites, with representative homology sequence color coded on blue DNA strand. b Genotyping for NHEJ events utilizes a three-primer system, with P1 being shared between P2 and P3. Primers P1 and P3 reside between 100 to 200 bp outside of the target site (an average deletion product size is depicted). The P1 + P3 primer pair may not always amplify a wild-type product, if the target sequences are too far apart. 1400 bp represents the average distance between loxP insertion sites. c Random ODN insertion PCR primers reside internal to homology arm sequence, and will amplify the expected size product if the ssODN donor has been incorporated elsewhere in the genome, away from the critical exon, in addition to the on-target locus. d Primers for the homology arm screen were used in a SYBR-green quantitative PCR reaction from DNA samples from the N1 generation, using β-actin as a two-copy normalization control

    Techniques Used: Non-Homologous End Joining, Polymerase Chain Reaction, Sequencing, SYBR Green Assay, Real-time Polymerase Chain Reaction

    29) Product Images from "Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae"

    Article Title: Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae

    Journal: Frontiers in Public Health

    doi: 10.3389/fpubh.2017.00109

    Agarose gel electrophoresis of SYBR green PCR products . Lanes 1 and 2 (10 3 gene copy): Vibrio cholerae O1 ATCC N16961 and V. cholerae O139 ATCC NIHC0270, respectively; lanes 3 and 4 (10 4 copies): V. cholerae O1 ATCC N16961 and V. cholerae O139 NIHC0270 ATCC, respectively; lanes 5 and 6 (10 5 copies): V. cholerae O1 ATCC N16961 and V. cholerae O139NIHC0270ATCC, respectively; lanes 7 and 8 (10 6 copies): V. cholerae O1 ATCC N16961 and V. cholerae O139NIHC0270ATCC, respectively; lanes 9 and 10 (10 7 copies): V. cholerae O1 ATCC N16961 and V. cholerae O139NIHC0270ATCC, respectively; Lane 10 (M): molecular weight marker (100 bp DNA Ladder, Karl Roth, Germany), 11 no template control.
    Figure Legend Snippet: Agarose gel electrophoresis of SYBR green PCR products . Lanes 1 and 2 (10 3 gene copy): Vibrio cholerae O1 ATCC N16961 and V. cholerae O139 ATCC NIHC0270, respectively; lanes 3 and 4 (10 4 copies): V. cholerae O1 ATCC N16961 and V. cholerae O139 NIHC0270 ATCC, respectively; lanes 5 and 6 (10 5 copies): V. cholerae O1 ATCC N16961 and V. cholerae O139NIHC0270ATCC, respectively; lanes 7 and 8 (10 6 copies): V. cholerae O1 ATCC N16961 and V. cholerae O139NIHC0270ATCC, respectively; lanes 9 and 10 (10 7 copies): V. cholerae O1 ATCC N16961 and V. cholerae O139NIHC0270ATCC, respectively; Lane 10 (M): molecular weight marker (100 bp DNA Ladder, Karl Roth, Germany), 11 no template control.

    Techniques Used: Agarose Gel Electrophoresis, SYBR Green Assay, Polymerase Chain Reaction, Molecular Weight, Marker

    Melt curve of SYBR green PCR products . The Y -axis represents the derivative reporter (−Rn) while x -axis represents the temperature (°C). The figure shows a melting temperature ( 31 ) of human ompW PCR products as 78.46°C.
    Figure Legend Snippet: Melt curve of SYBR green PCR products . The Y -axis represents the derivative reporter (−Rn) while x -axis represents the temperature (°C). The figure shows a melting temperature ( 31 ) of human ompW PCR products as 78.46°C.

    Techniques Used: SYBR Green Assay, Polymerase Chain Reaction

    30) Product Images from "Deregulation of CREB Signaling Pathway Induced by Chronic Hyperglycemia Downregulates NeuroD Transcription"

    Article Title: Deregulation of CREB Signaling Pathway Induced by Chronic Hyperglycemia Downregulates NeuroD Transcription

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034860

    Chronic hyperglycemia alters the responsiveness to cAMP in pancreatic islets. ( A ) Rat islet cells were cultured in the presence of 5 mM or 30 mM glucose for 8 days and stimulated with 30 µM forskolin for 0–12 h after 2 h preconditioning in 5 mM glucose. ( B∼F ) Real-time PCR was carried out using SYBR green to quantitate the mRNA levels of indicated genes in diverse conditions shown in A . Relative mRNA levels were estimated from Ct values summarized in Supporting Table S1 using 2 −ΔΔCt method. The data from three independent experiments are presented as average fold ratios of relative mRNA expression compare with the 5 mM glucose-cultured islets before forskolin treatment in 5 mM glucose-cultured islets. Significant effects of forskolin (*, P
    Figure Legend Snippet: Chronic hyperglycemia alters the responsiveness to cAMP in pancreatic islets. ( A ) Rat islet cells were cultured in the presence of 5 mM or 30 mM glucose for 8 days and stimulated with 30 µM forskolin for 0–12 h after 2 h preconditioning in 5 mM glucose. ( B∼F ) Real-time PCR was carried out using SYBR green to quantitate the mRNA levels of indicated genes in diverse conditions shown in A . Relative mRNA levels were estimated from Ct values summarized in Supporting Table S1 using 2 −ΔΔCt method. The data from three independent experiments are presented as average fold ratios of relative mRNA expression compare with the 5 mM glucose-cultured islets before forskolin treatment in 5 mM glucose-cultured islets. Significant effects of forskolin (*, P

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, SYBR Green Assay, Expressing

    Chronic hyperglycemia alters the β-cell specific gene expression in pancreatic islets. ( A ) Rat islet cells were cultured in the presence of 5 mM or 30 mM glucose for 8 days, and stimulated with 15 mM glucose for 0–6 h after 2 h preconditioning in 5 mM glucose. ( B ) Dithizone staining of islet cells after 8 days of culture. Scale bar: 100 µm. ( C ) In low (5 mM) glucose condition, insulin secretion was normal in response to acute stimulation with 15 mM glucose after 8 day culture whereas insulin secretion was impaired after 8-day exposure to high (30 mM) glucose. Under high glucose conditions, cellular insulin content were significantly reduced, and total proteins were decreased slightly. ( D∼H ) Real-time PCR was carried out using SYBR green to quantitate the mRNA levels of indicated genes in diverse conditions shown in A . Relative mRNA levels were estimated from of Ct values summarized in Supporting Table S1 using 2 −ΔΔCt method. The data from three independent experiments are presented as average fold ratios (means ± S.E.) of relative mRNA expression compare with the 5 mM glucose-cultured islets before glucose stimulation. Simultaneous decreases in the mRNA levels and the intracellular insulin content correlated well. Significant effects of 15 mM glucose (*, P
    Figure Legend Snippet: Chronic hyperglycemia alters the β-cell specific gene expression in pancreatic islets. ( A ) Rat islet cells were cultured in the presence of 5 mM or 30 mM glucose for 8 days, and stimulated with 15 mM glucose for 0–6 h after 2 h preconditioning in 5 mM glucose. ( B ) Dithizone staining of islet cells after 8 days of culture. Scale bar: 100 µm. ( C ) In low (5 mM) glucose condition, insulin secretion was normal in response to acute stimulation with 15 mM glucose after 8 day culture whereas insulin secretion was impaired after 8-day exposure to high (30 mM) glucose. Under high glucose conditions, cellular insulin content were significantly reduced, and total proteins were decreased slightly. ( D∼H ) Real-time PCR was carried out using SYBR green to quantitate the mRNA levels of indicated genes in diverse conditions shown in A . Relative mRNA levels were estimated from of Ct values summarized in Supporting Table S1 using 2 −ΔΔCt method. The data from three independent experiments are presented as average fold ratios (means ± S.E.) of relative mRNA expression compare with the 5 mM glucose-cultured islets before glucose stimulation. Simultaneous decreases in the mRNA levels and the intracellular insulin content correlated well. Significant effects of 15 mM glucose (*, P

    Techniques Used: Expressing, Cell Culture, Staining, Real-time Polymerase Chain Reaction, SYBR Green Assay

    31) Product Images from "Trematode Fluke Procerovumvarium as Cause of Ocular Inflammation in Children, South India"

    Article Title: Trematode Fluke Procerovumvarium as Cause of Ocular Inflammation in Children, South India

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2202.150051

    Real-time PCR amplification of ocular granuloma DNA obtained from patients infected with trematodes, South India. Gel electrophoresis was performed on 2% agarose gel by using Power SYBR Green Real-Time PCR (Applied Biosystems, Warrington, UK). A) Lanes 1–4 show subconjunctival granuloma DNA; lane 5, negative control; lane 6, 100-bp DNA marker. Arrow indicates 369-bp amplified DNA product. B) Lanes 1–5 show anterior chamber granuloma DNA; lane 6, negative control; lane 7, 100-bp DNA marker. Arrow indicates 369-bp amplified DNA product. C) BLAST ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) analysis output of patient granuloma DNA sequence showing maximum identity with internal transcribed spacer 2 region gene sequence of the Procerovum species resembling GenBank reported sequence EU826639.1 from Vietnam.
    Figure Legend Snippet: Real-time PCR amplification of ocular granuloma DNA obtained from patients infected with trematodes, South India. Gel electrophoresis was performed on 2% agarose gel by using Power SYBR Green Real-Time PCR (Applied Biosystems, Warrington, UK). A) Lanes 1–4 show subconjunctival granuloma DNA; lane 5, negative control; lane 6, 100-bp DNA marker. Arrow indicates 369-bp amplified DNA product. B) Lanes 1–5 show anterior chamber granuloma DNA; lane 6, negative control; lane 7, 100-bp DNA marker. Arrow indicates 369-bp amplified DNA product. C) BLAST ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) analysis output of patient granuloma DNA sequence showing maximum identity with internal transcribed spacer 2 region gene sequence of the Procerovum species resembling GenBank reported sequence EU826639.1 from Vietnam.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Infection, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, SYBR Green Assay, Negative Control, Marker, Sequencing

    32) Product Images from "Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles"

    Article Title: Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles

    Journal: BMC Biology

    doi: 10.1186/s12915-018-0529-0

    Screening strategies for HDR and NHEJ alleles, and random ODN insertion. Relative positions of the primers and approximate sizes of PCR products are listed below each allele. Scissors represent target sites. a Genotyping schemes for detecting loxP donor sequences. Orange triangles represent loxP sites, with representative homology sequence color coded on blue DNA strand. b Genotyping for NHEJ events utilizes a three-primer system, with P1 being shared between P2 and P3. Primers P1 and P3 reside between 100 to 200 bp outside of the target site (an average deletion product size is depicted). The P1 + P3 primer pair may not always amplify a wild-type product, if the target sequences are too far apart. 1400 bp represents the average distance between loxP insertion sites. c Random ODN insertion PCR primers reside internal to homology arm sequence, and will amplify the expected size product if the ssODN donor has been incorporated elsewhere in the genome, away from the critical exon, in addition to the on-target locus. d Primers for the homology arm screen were used in a SYBR-green quantitative PCR reaction from DNA samples from the N1 generation, using β-actin as a two-copy normalization control
    Figure Legend Snippet: Screening strategies for HDR and NHEJ alleles, and random ODN insertion. Relative positions of the primers and approximate sizes of PCR products are listed below each allele. Scissors represent target sites. a Genotyping schemes for detecting loxP donor sequences. Orange triangles represent loxP sites, with representative homology sequence color coded on blue DNA strand. b Genotyping for NHEJ events utilizes a three-primer system, with P1 being shared between P2 and P3. Primers P1 and P3 reside between 100 to 200 bp outside of the target site (an average deletion product size is depicted). The P1 + P3 primer pair may not always amplify a wild-type product, if the target sequences are too far apart. 1400 bp represents the average distance between loxP insertion sites. c Random ODN insertion PCR primers reside internal to homology arm sequence, and will amplify the expected size product if the ssODN donor has been incorporated elsewhere in the genome, away from the critical exon, in addition to the on-target locus. d Primers for the homology arm screen were used in a SYBR-green quantitative PCR reaction from DNA samples from the N1 generation, using β-actin as a two-copy normalization control

    Techniques Used: Non-Homologous End Joining, Polymerase Chain Reaction, Sequencing, SYBR Green Assay, Real-time Polymerase Chain Reaction

    33) Product Images from "The Flavones Apigenin and Luteolin Induce FOXO1 Translocation but Inhibit Gluconeogenic and Lipogenic Gene Expression in Human Cells"

    Article Title: The Flavones Apigenin and Luteolin Induce FOXO1 Translocation but Inhibit Gluconeogenic and Lipogenic Gene Expression in Human Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0104321

    Time- and dose-dependent modulation of gene expression in HepG2 cells induced by apigenin and luteolin. A–D: Human hepatoma cells (HepG2) were cultivated in EMEM + 10% FBS and starved without FBS 16 h before stimulation. Apigenin and luteolin were applied in the range of 1–100 µM diluted in EMEM. Incubation of HepG2 was performed for 2 h and 24 h respectively. Total RNA was extracted with Nucleospin RNA II isolation kit and reverse transcribed with the High capacity cDNA reverse transcription kit for quantitative realtime PCR (qRT-PCR) in triplicates using the Power SYBR green PCR master mix with primers pairs described in Table 1 . qRT-PCR was run in triplicates using cDNA from control cells treated with DMSO 0.5% for standard dilutions. Levels of mRNA were normalized to the houskeeping gene ribosomal protein (RPL32). Three independent experiments were performed with different passages of HepG2. Results are presented as fold mRNA expression normalized to control expression as means ± SEM and significances versus control *(p
    Figure Legend Snippet: Time- and dose-dependent modulation of gene expression in HepG2 cells induced by apigenin and luteolin. A–D: Human hepatoma cells (HepG2) were cultivated in EMEM + 10% FBS and starved without FBS 16 h before stimulation. Apigenin and luteolin were applied in the range of 1–100 µM diluted in EMEM. Incubation of HepG2 was performed for 2 h and 24 h respectively. Total RNA was extracted with Nucleospin RNA II isolation kit and reverse transcribed with the High capacity cDNA reverse transcription kit for quantitative realtime PCR (qRT-PCR) in triplicates using the Power SYBR green PCR master mix with primers pairs described in Table 1 . qRT-PCR was run in triplicates using cDNA from control cells treated with DMSO 0.5% for standard dilutions. Levels of mRNA were normalized to the houskeeping gene ribosomal protein (RPL32). Three independent experiments were performed with different passages of HepG2. Results are presented as fold mRNA expression normalized to control expression as means ± SEM and significances versus control *(p

    Techniques Used: Expressing, Incubation, Isolation, Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay

    Gene expression profiling upon siRNA knockdowns and analysis of modulations by apigenin and luteolin. A–D: Subconfluent human hepatoma cells (HepG2) were transfected with silencing RNA (siRNA) for forkhead box transcription factor O1 (FOXO1), forkhead box transcription factor O3a (FOXO3a), sirtuin1 (SIRT1), protein kinase B (PKB/AKT), nuclear factor (erythroid-derived2)-like2 (NRF2) and non targeting (NT)-siRNA with DharmaFECT4 in EMEM + 10% FBS for 48 h including a starvation period without FBS of 16 h preceeding stimulation with apigenin and luteolin each 20 µM for 24 h. RNA was extracted, reverse transcribed and cDNA from control cells after treatment with DMSO 0.1% were used for standard dilutions. For 14 targets qRT-PCR was run with SYBR green in triplicates. Levels of mRNA were normalized to the expression of houskeeping ribosomal protein (RPL32) mRNA. At least four experiments (n = 4–8) transfecting each siRNA or combined siRNAs for single and double knockdowns and control transfections with NT-siRNA followed by apigenin, luteolin or mock treatment with DMSO 0.1% were performed with different passages of HepG2. Ratios of mRNA levels vs basal expression in NT-siRNA transfected cells were calculated for knockdown induced fold mRNA of basal levels (grey columns). T-tests were performed for independent samples and significances versus control are shown # p
    Figure Legend Snippet: Gene expression profiling upon siRNA knockdowns and analysis of modulations by apigenin and luteolin. A–D: Subconfluent human hepatoma cells (HepG2) were transfected with silencing RNA (siRNA) for forkhead box transcription factor O1 (FOXO1), forkhead box transcription factor O3a (FOXO3a), sirtuin1 (SIRT1), protein kinase B (PKB/AKT), nuclear factor (erythroid-derived2)-like2 (NRF2) and non targeting (NT)-siRNA with DharmaFECT4 in EMEM + 10% FBS for 48 h including a starvation period without FBS of 16 h preceeding stimulation with apigenin and luteolin each 20 µM for 24 h. RNA was extracted, reverse transcribed and cDNA from control cells after treatment with DMSO 0.1% were used for standard dilutions. For 14 targets qRT-PCR was run with SYBR green in triplicates. Levels of mRNA were normalized to the expression of houskeeping ribosomal protein (RPL32) mRNA. At least four experiments (n = 4–8) transfecting each siRNA or combined siRNAs for single and double knockdowns and control transfections with NT-siRNA followed by apigenin, luteolin or mock treatment with DMSO 0.1% were performed with different passages of HepG2. Ratios of mRNA levels vs basal expression in NT-siRNA transfected cells were calculated for knockdown induced fold mRNA of basal levels (grey columns). T-tests were performed for independent samples and significances versus control are shown # p

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, SYBR Green Assay

    FOXO targetgene-expression in HepG2 (human hepatoma) cells modulated by polyphenolic resveratrol in a time-dependent course. A–B: HepG2 cell cultures grown in EMEM + FBS 10% and starved for 16 h without FBS were stimulated with resveratrol 50 µM in 0.125% DMSO in EMEM for 1–24 h. RNA was extracted with Nucleospin RNA II isolation kit and reverse transcribed with the High capacity cDNA reverse transcription kit for quantitative realtime PCR (qRT-PCR) in triplicates using the Power SYBR green PCR master mix with primers pairs described in Table 1 . Modulated mRNA levels normalized to ribosomal protein (RPL32) housekeeping gene are shown as fold mRNA of basal expression in mock stimulated HepG2 means ± SEM (n = 3) of 3 independent experiments with different passages of HepG2 with significances (t-test) versus DMSO-control *p
    Figure Legend Snippet: FOXO targetgene-expression in HepG2 (human hepatoma) cells modulated by polyphenolic resveratrol in a time-dependent course. A–B: HepG2 cell cultures grown in EMEM + FBS 10% and starved for 16 h without FBS were stimulated with resveratrol 50 µM in 0.125% DMSO in EMEM for 1–24 h. RNA was extracted with Nucleospin RNA II isolation kit and reverse transcribed with the High capacity cDNA reverse transcription kit for quantitative realtime PCR (qRT-PCR) in triplicates using the Power SYBR green PCR master mix with primers pairs described in Table 1 . Modulated mRNA levels normalized to ribosomal protein (RPL32) housekeeping gene are shown as fold mRNA of basal expression in mock stimulated HepG2 means ± SEM (n = 3) of 3 independent experiments with different passages of HepG2 with significances (t-test) versus DMSO-control *p

    Techniques Used: Expressing, Isolation, Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay, T-Test

    34) Product Images from "High Yield Production of Influenza Virus in Madin Darby Canine Kidney (MDCK) Cells with Stable Knockdown of IRF7"

    Article Title: High Yield Production of Influenza Virus in Madin Darby Canine Kidney (MDCK) Cells with Stable Knockdown of IRF7

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059892

    Knockdown of IRF7 enhances PR8 virus production in MDCK cells. (A) MDCK cells were transfected with siRNAs targeting the indicated genes. At 48 h post-transfection, the cells were infected with PR8 virus at a MOI of 0.01. After 24 h of incubation, viral RNA in culture supernatant was measured by quantitative real-time RT-PCR. The gray boxes in the panel mean that siRNA which targets indicated gene showed more than 2-fold enhancement of the virus production compared with negative control siRNA. (B) Total RNA from cells was extracted to monitor knockdown efficiency of target gene in the cells. The endogenous expression of target genes was measured by quantitative real-time RT-PCR with SYBR green. (C) MDCK cells were transfected with three siRNAs targeting genes or negative control siRNA. At 48 h post-transfection, the cells were infected with PR8 virus at a MOI of 0.01. After 24 h of incubation, the RNA from culture supernatant was isolated to examine the amounts of a specific viral RNA. The data are representative results of three independent experiments. Asterisks indicate statistically significant differences compared with the control (*P
    Figure Legend Snippet: Knockdown of IRF7 enhances PR8 virus production in MDCK cells. (A) MDCK cells were transfected with siRNAs targeting the indicated genes. At 48 h post-transfection, the cells were infected with PR8 virus at a MOI of 0.01. After 24 h of incubation, viral RNA in culture supernatant was measured by quantitative real-time RT-PCR. The gray boxes in the panel mean that siRNA which targets indicated gene showed more than 2-fold enhancement of the virus production compared with negative control siRNA. (B) Total RNA from cells was extracted to monitor knockdown efficiency of target gene in the cells. The endogenous expression of target genes was measured by quantitative real-time RT-PCR with SYBR green. (C) MDCK cells were transfected with three siRNAs targeting genes or negative control siRNA. At 48 h post-transfection, the cells were infected with PR8 virus at a MOI of 0.01. After 24 h of incubation, the RNA from culture supernatant was isolated to examine the amounts of a specific viral RNA. The data are representative results of three independent experiments. Asterisks indicate statistically significant differences compared with the control (*P

    Techniques Used: Transfection, Infection, Incubation, Quantitative RT-PCR, Negative Control, Expressing, SYBR Green Assay, Isolation

    35) Product Images from "The Human Hyaluronan Synthase 2 (HAS2) Gene and Its Natural Antisense RNA Exhibit Coordinated Expression in the Renal Proximal Tubular Epithelial Cell ♦"

    Article Title: The Human Hyaluronan Synthase 2 (HAS2) Gene and Its Natural Antisense RNA Exhibit Coordinated Expression in the Renal Proximal Tubular Epithelial Cell ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.233916

    End-point RT-PCR detection of HAS2-AS1 transcription, optimization of SYBR Green qRT-PCR, and absolute quantification of HAS2 and HAS2-AS1 RNAs. A , detection of HAS2-AS1 expression in PTCs by end-point RT-PCR using primers A and AR (see B for details
    Figure Legend Snippet: End-point RT-PCR detection of HAS2-AS1 transcription, optimization of SYBR Green qRT-PCR, and absolute quantification of HAS2 and HAS2-AS1 RNAs. A , detection of HAS2-AS1 expression in PTCs by end-point RT-PCR using primers A and AR (see B for details

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Quantitative RT-PCR, Expressing

    36) Product Images from "Knockdown of CypA inhibits interleukin-8 (IL-8) and IL-8-mediated proliferation and tumor growth of glioblastoma cells through down-regulated NF-?B"

    Article Title: Knockdown of CypA inhibits interleukin-8 (IL-8) and IL-8-mediated proliferation and tumor growth of glioblastoma cells through down-regulated NF-?B

    Journal: Journal of Neuro-Oncology

    doi: 10.1007/s11060-010-0220-y

    CypA KD-mediated down-regulation of IL-8 expression. a IL - 8 expression levels were determined by a quantitative real-time PCR analysis. One microgram of total RNA was used for cDNA synthesis, using Superscript III (Invitrogen) with the oligo(dT) 15 primer. Quantitative real-time PCR (QRT-PCR) was performed using the Power SYBR Green PCR Master Mix (Applied Biosystem) detected by the 7300 Real Time PCR System (Applied Biosystems). The relative expression of IL - 8 mRNA normalized to the internal reference 18S rRNA was analyzed using the 2 −ΔΔCT method ( columns , mean of results from three independent experiments; bars, SE; *, P
    Figure Legend Snippet: CypA KD-mediated down-regulation of IL-8 expression. a IL - 8 expression levels were determined by a quantitative real-time PCR analysis. One microgram of total RNA was used for cDNA synthesis, using Superscript III (Invitrogen) with the oligo(dT) 15 primer. Quantitative real-time PCR (QRT-PCR) was performed using the Power SYBR Green PCR Master Mix (Applied Biosystem) detected by the 7300 Real Time PCR System (Applied Biosystems). The relative expression of IL - 8 mRNA normalized to the internal reference 18S rRNA was analyzed using the 2 −ΔΔCT method ( columns , mean of results from three independent experiments; bars, SE; *, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay, Polymerase Chain Reaction

    37) Product Images from "Targeting levels or oligomerization of nucleophosmin 1 induces differentiation and loss of survival of human AML cells with mutant NPM1"

    Article Title: Targeting levels or oligomerization of nucleophosmin 1 induces differentiation and loss of survival of human AML cells with mutant NPM1

    Journal: Blood

    doi: 10.1182/blood-2010-09-309674

    Depletion of NPM1 down-regulates the expression of leukemogenic markers induces G0/G1 accumulation and markedly reduces clonogenic survival of AML cells. (A) Top panel, HL-60 and OCI-AML3 cells were transfected with control siRNA or NPM1 siRNA for 24 hours. After 24 hours, total RNA was extracted and semi-quantitative RT-PCR was performed for WT-NPM1, Mt-NPM1, HOXA9, Meis1, and FLT3. The levels of β-actin mRNA served as the loading control. Bottom panel, quantitative PCR reactions were also performed with SYBR Green to assess NPM1 depletion in the siRNA transfected cells. Relative expression of NPM-1 was normalized to GAPDH. (B) HL-60 and OCI-AML3 cells were transfected with control (−) or NPM1 (+) siRNA and incubated for 48 hours. At the end of incubation, nuclear and cytosolic fractions were isolated and immunoblot analyses were performed for NPM1, HOXA9, Meis1, and FLT3. Expression levels of β-actin and EZH2 served as the loading and fraction controls for the cytosolic and nuclear extracts, respectively. (C) HL-60 and OCI-AML3 cells were transfected with control or NPM1-siRNA and incubated for 96 hours. Then, cells were fixed and stained with propidium iodide and cell cycle status was determined by flow cytometry. Values represent the mean of 3 independent experiments + SEM. (+) indicates G0/G1 values significantly different ( P
    Figure Legend Snippet: Depletion of NPM1 down-regulates the expression of leukemogenic markers induces G0/G1 accumulation and markedly reduces clonogenic survival of AML cells. (A) Top panel, HL-60 and OCI-AML3 cells were transfected with control siRNA or NPM1 siRNA for 24 hours. After 24 hours, total RNA was extracted and semi-quantitative RT-PCR was performed for WT-NPM1, Mt-NPM1, HOXA9, Meis1, and FLT3. The levels of β-actin mRNA served as the loading control. Bottom panel, quantitative PCR reactions were also performed with SYBR Green to assess NPM1 depletion in the siRNA transfected cells. Relative expression of NPM-1 was normalized to GAPDH. (B) HL-60 and OCI-AML3 cells were transfected with control (−) or NPM1 (+) siRNA and incubated for 48 hours. At the end of incubation, nuclear and cytosolic fractions were isolated and immunoblot analyses were performed for NPM1, HOXA9, Meis1, and FLT3. Expression levels of β-actin and EZH2 served as the loading and fraction controls for the cytosolic and nuclear extracts, respectively. (C) HL-60 and OCI-AML3 cells were transfected with control or NPM1-siRNA and incubated for 96 hours. Then, cells were fixed and stained with propidium iodide and cell cycle status was determined by flow cytometry. Values represent the mean of 3 independent experiments + SEM. (+) indicates G0/G1 values significantly different ( P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, SYBR Green Assay, Incubation, Isolation, Staining, Flow Cytometry, Cytometry

    38) Product Images from "An Optimized Real-Time PCR to Avoid Species-/Tissue-Associated Inhibition for H5N1 Detection in Ferret and Monkey Tissues"

    Article Title: An Optimized Real-Time PCR to Avoid Species-/Tissue-Associated Inhibition for H5N1 Detection in Ferret and Monkey Tissues

    Journal: The Scientific World Journal

    doi: 10.1100/2012/907095

    Agarose gel electrophoresis of SYBR Green real-time PCR products of H5N1 transcripts in lung and small intestine tissues. Lane 1: DL2000 Marker; Lane 2: positive control; Lanes 3–5: small intestine samples amplified with primer pairs N1-1/N1-2, M30F/M264R, H5-1/H5-3; Lanes 6–8: lung samples amplified with primer pairs N1-1/N1-2, M30F/M264R, H5-1/H5-3; Lanes 9–13: small intestine samples prepared by TR, QI, BI, AM, and RO, respectively, and amplified by primer pair SZNP-F2/R2; Lanes 14–18: lung samples prepared by TR, QI, BI, AM, and RO, respectively, and amplified with primer pair SZNP-F2/R2; Lane 19: negative control. (a) was for ferret tissues and (b) was for monkey tissues.
    Figure Legend Snippet: Agarose gel electrophoresis of SYBR Green real-time PCR products of H5N1 transcripts in lung and small intestine tissues. Lane 1: DL2000 Marker; Lane 2: positive control; Lanes 3–5: small intestine samples amplified with primer pairs N1-1/N1-2, M30F/M264R, H5-1/H5-3; Lanes 6–8: lung samples amplified with primer pairs N1-1/N1-2, M30F/M264R, H5-1/H5-3; Lanes 9–13: small intestine samples prepared by TR, QI, BI, AM, and RO, respectively, and amplified by primer pair SZNP-F2/R2; Lanes 14–18: lung samples prepared by TR, QI, BI, AM, and RO, respectively, and amplified with primer pair SZNP-F2/R2; Lane 19: negative control. (a) was for ferret tissues and (b) was for monkey tissues.

    Techniques Used: Agarose Gel Electrophoresis, SYBR Green Assay, Real-time Polymerase Chain Reaction, Marker, Positive Control, Amplification, Negative Control

    The sensitivity and specificity achieved with primer pair SZNP-F2/R2 in SYBR Green real-time PCR. (a) Standard curve. LogCO: log10 (copies), slope: −3.4779, R 2 : 0.998, eff%: 110%. (b) Melting curve. (c) Agarose electrophoresis of PCR products of H5N1. Lane 1: DL2000 Marker; Lanes 2–9: 10 8 –10 1 copies of H5N1 virus cDNA.
    Figure Legend Snippet: The sensitivity and specificity achieved with primer pair SZNP-F2/R2 in SYBR Green real-time PCR. (a) Standard curve. LogCO: log10 (copies), slope: −3.4779, R 2 : 0.998, eff%: 110%. (b) Melting curve. (c) Agarose electrophoresis of PCR products of H5N1. Lane 1: DL2000 Marker; Lanes 2–9: 10 8 –10 1 copies of H5N1 virus cDNA.

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction, Electrophoresis, Polymerase Chain Reaction, Marker

    39) Product Images from "Impact of lipoteichoic acid modification on the performance of the probiotic Lactobacillus rhamnosus GG in experimental colitis"

    Article Title: Impact of lipoteichoic acid modification on the performance of the probiotic Lactobacillus rhamnosus GG in experimental colitis

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/j.1365-2249.2010.04228.x

    Cytokine quantification in the colon of dextran sulphate sodium (DSS)-induced colitis mice (1% DSS model). Mice were given phosphate-buffered saline (PBS), Lactobacillus rhamnosus GG (LGG) wild-type or dltD mutant and killed at day 43 after induction of colitis. Interleukin (IL)-12p40 (a), interferon (IFN)-γ (b), Toll-like receptor (TLR)-2 (c), TLR-1 (d), TLR-4 (e) and TLR-6 (f) were quantified by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) as described in Materials and methods. The expression was normalized against the housekeeping gene β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). IL-12p40 and IFN-γ were analysed with Taq Man probes and TLR-expression was measured by SYBR Green (2 -ΔΔCt method). Data represent mean values ± standard error of the mean.
    Figure Legend Snippet: Cytokine quantification in the colon of dextran sulphate sodium (DSS)-induced colitis mice (1% DSS model). Mice were given phosphate-buffered saline (PBS), Lactobacillus rhamnosus GG (LGG) wild-type or dltD mutant and killed at day 43 after induction of colitis. Interleukin (IL)-12p40 (a), interferon (IFN)-γ (b), Toll-like receptor (TLR)-2 (c), TLR-1 (d), TLR-4 (e) and TLR-6 (f) were quantified by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) as described in Materials and methods. The expression was normalized against the housekeeping gene β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). IL-12p40 and IFN-γ were analysed with Taq Man probes and TLR-expression was measured by SYBR Green (2 -ΔΔCt method). Data represent mean values ± standard error of the mean.

    Techniques Used: Mouse Assay, Mutagenesis, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, SYBR Green Assay

    40) Product Images from "Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F"

    Article Title: Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F

    Journal: Journal of Virology

    doi: 10.1128/JVI.01101-17

    qRT-PCR analyses of HPIV1 F and HN RNA. Vero cells were infected with the indicated viruses at an MOI of 5 TCID 50 per cell at 32°C for 48 h. Total RNA was extracted from infected cells and reverse transcribed into cDNA. The transcription levels of HPIV1 F (A) and HN (B) were quantified using Power SYBR green PCR master mix according to the manufacturer's instruction. GAPDH was used as an endogenous control. All samples were analyzed in triplicate and the results are shown as mean level of transcripts ± the SEM relative to rHPIV1-C Δ170 empty vector, whose expression is set at 100%.
    Figure Legend Snippet: qRT-PCR analyses of HPIV1 F and HN RNA. Vero cells were infected with the indicated viruses at an MOI of 5 TCID 50 per cell at 32°C for 48 h. Total RNA was extracted from infected cells and reverse transcribed into cDNA. The transcription levels of HPIV1 F (A) and HN (B) were quantified using Power SYBR green PCR master mix according to the manufacturer's instruction. GAPDH was used as an endogenous control. All samples were analyzed in triplicate and the results are shown as mean level of transcripts ± the SEM relative to rHPIV1-C Δ170 empty vector, whose expression is set at 100%.

    Techniques Used: Quantitative RT-PCR, Infection, SYBR Green Assay, Polymerase Chain Reaction, Plasmid Preparation, Expressing

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    Real-time Polymerase Chain Reaction:

    Article Title: Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis
    Article Snippet: .. Aliquots (12.5 ng) of cDNA were amplified in triplicate using Power SYBR green PCR master mix (Thermo Fisher Scientific) and Qiagen QuantiTect primer assays for human hypoxanthine phosphoribosyltransferase (HPRT) (QT00059066), APOBEC3G (QT00070770), SLC20A1 (PIT-1) (QT00028763), or SLC19A2 (THTR1) (QT00007847) on an ABI 7500 real-time PCR system. .. Quantification was carried out relative to HEK293 cells, and values were normalized to HPRT expression.

    Article Title: Increased expression of readthrough acetylcholinesterase variants in the brains of Alzheimer's disease patients
    Article Snippet: .. First-strand cDNAs were synthesized by reverse transcription of 1.5 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Life Technologies Paisley, UK), according to the manufacturer's instructions. q RT-PCR amplification was performed using StepOne-Plus™ Real-Time PCR System with Power SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions for analysis of AChE transcripts. .. GAPDH mRNA was used as the housekeeping marker.

    Article Title: Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells
    Article Snippet: .. For mRNA analysis, total RNA was extracted from cells by TRIzol (Invitrogen) according to the manufacturer’s protocol and reverse-transcribed into cDNA using the QuantiTect® reverse transcription kit. cDNA was quantitated using Power SYBR Green PCR (Applied Biosystems by Life Technologies, Warrington, UK) and the QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems). ..

    Article Title: Estradiol Protects Proopiomelanocortin Neurons Against Insulin Resistance
    Article Snippet: .. The results were as follows: Pik3cb (p110 β ), m = –3.481, r 2 = 0.95, efficiency = 96%; Gapdh , m = –3.35, r 2 = 0.99, efficiency = 98.7%; Socs3 , m = –3.294, r 2 = 0.90, efficiency = 100%; Trpc5 , m = –3.161, r 2 = 0.95, efficiency = 100%; Stim1 , m = –3.407, r 2 = 0.98, efficiency = 97%. qPCR was performed on a Quantstudio 7 Flex Real-Time PCR System (Life Technologies, Grand Island, NY) using Power SYBR Green Master Mix (Life Technologies) according to established protocols ( ). ..

    Article Title: secA, secD, secF, yajC, and yidC contribute to the adhesion regulation of Vibrio alginolyticus. secA, secD, secF, yajC, and yidC contribute to the adhesion regulation of Vibrio alginolyticus
    Article Snippet: .. 2.5 Quantitative real‐time PCR (qRT‐PCR) Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, California, USA) was used to assay the expression of genes by qRT‐PCR according to the manufacturer's instructions. .. 16s RNA was used to normalize the expression levels.

    Article Title: Regulation of ?4-integrin expression by epigenetic modifications in the mammary gland and during the epithelial-to-mesenchymal transition
    Article Snippet: .. For quantitative RT-PCR (qRT-PCR), reverse-transcribed cDNA was used for real-time PCR using Power SYBR Green Master Mix (Applied Biosystems) with the HT7900 Fast Real Time PCR machine (Applied Biosystems). .. Standard curves were generated using serial dilutions of cDNA with arbitrary copy numbers.

    Polymerase Chain Reaction:

    Article Title: Cyclophilin A (CypA) Interacts with NF-κB Subunit, p65/RelA, and Contributes to NF-κB Activation Signaling
    Article Snippet: .. Using the cDNA synthesized from the SuperScript III First-Strand Synthesis System as templates, individual specific primers (p65 F, 5′-GACGACTGTTCCCCCTC-3′ and R, 5′-CCTCGCACTTGTAGCGG-3′ ; p50 F, 5′-GCAAACCTGGGAATACTTCATGTGACTAAG-3′ and R, 5′-ATAGGCAA-CCTCAGAATGCACCAGAAGTCC-3′ ; IL-1β F, 5′- TGCGTGTTGAAAGATGATAA-G-3′ and R, 5′- TTGGGGAACTGGGCAGAC-3′ ; IL-6 F, 5′-CTCCAGAACAGATTT-GAGAGTAGTG-3′ and R, 5′-TTGTGGTTGGGTCAGGGGTG-3′ ; IL-8 F, 5′-GGGTT-GTGGAGAAGTTTTTG-3′ and R, 5′-GTTTCACTGGCATCTTCACTG-3′ ) were added to each template and mixed with the Power SYBR Green PCR Master Mix (Life Technologies, Gaitherburg, MD). .. The reactions were performed using the 7300 Real-Time PCR System instrument (Applied Biosystems, Foster City, CA).

    Article Title: CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells
    Article Snippet: .. Equal volumes of prepared cDNA were used for quantification of HSV-1 gene ICP0 and ICP4 transcripts with the Power SYBR Green PCR master mix (Applied biosystems) according to the manufacturer's protocol. ..

    Article Title: Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis
    Article Snippet: .. Aliquots (12.5 ng) of cDNA were amplified in triplicate using Power SYBR green PCR master mix (Thermo Fisher Scientific) and Qiagen QuantiTect primer assays for human hypoxanthine phosphoribosyltransferase (HPRT) (QT00059066), APOBEC3G (QT00070770), SLC20A1 (PIT-1) (QT00028763), or SLC19A2 (THTR1) (QT00007847) on an ABI 7500 real-time PCR system. .. Quantification was carried out relative to HEK293 cells, and values were normalized to HPRT expression.

    Article Title: Increased expression of readthrough acetylcholinesterase variants in the brains of Alzheimer's disease patients
    Article Snippet: .. First-strand cDNAs were synthesized by reverse transcription of 1.5 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Life Technologies Paisley, UK), according to the manufacturer's instructions. q RT-PCR amplification was performed using StepOne-Plus™ Real-Time PCR System with Power SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions for analysis of AChE transcripts. .. GAPDH mRNA was used as the housekeeping marker.

    Article Title: Role of miRNA-181a-2-3p in cadmium-induced inflammatory responses of human bronchial epithelial cells
    Article Snippet: .. For mRNA analysis, total RNA was extracted from cells by TRIzol (Invitrogen) according to the manufacturer’s protocol and reverse-transcribed into cDNA using the QuantiTect® reverse transcription kit. cDNA was quantitated using Power SYBR Green PCR (Applied Biosystems by Life Technologies, Warrington, UK) and the QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems). ..

    Article Title: secA, secD, secF, yajC, and yidC contribute to the adhesion regulation of Vibrio alginolyticus. secA, secD, secF, yajC, and yidC contribute to the adhesion regulation of Vibrio alginolyticus
    Article Snippet: .. 2.5 Quantitative real‐time PCR (qRT‐PCR) Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, California, USA) was used to assay the expression of genes by qRT‐PCR according to the manufacturer's instructions. .. 16s RNA was used to normalize the expression levels.

    Expressing:

    Article Title: secA, secD, secF, yajC, and yidC contribute to the adhesion regulation of Vibrio alginolyticus. secA, secD, secF, yajC, and yidC contribute to the adhesion regulation of Vibrio alginolyticus
    Article Snippet: .. 2.5 Quantitative real‐time PCR (qRT‐PCR) Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, California, USA) was used to assay the expression of genes by qRT‐PCR according to the manufacturer's instructions. .. 16s RNA was used to normalize the expression levels.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Increased expression of readthrough acetylcholinesterase variants in the brains of Alzheimer's disease patients
    Article Snippet: .. First-strand cDNAs were synthesized by reverse transcription of 1.5 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Life Technologies Paisley, UK), according to the manufacturer's instructions. q RT-PCR amplification was performed using StepOne-Plus™ Real-Time PCR System with Power SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions for analysis of AChE transcripts. .. GAPDH mRNA was used as the housekeeping marker.

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  • 99
    Thermo Fisher power sybr green pcr master mix
    Hypermutation of the FeLV-B genome and APOBEC3G mRNA expression in human cells. FeLV genomic sequences (segments of Gag and Pol) were cloned by <t>PCR</t> from infected human cells, and individual templates were sequenced and compared to the reference input virus (pFGB clone). APOBEC3G mRNA expression was determined in the same cells (prior to infection) by quantitative real-time PCR (with <t>SYBR</t> green). (A) Representative plots of hypermutation visualized by the online HYPERMUT program ( www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html ), where sequence changes relative to the reference FeLV-B genome are color coded (red, GG→AG; cyan, GA→AA; green, GC→AC; magenta, GT→AT; black, non-G→A). (B) X/Y plots of G→A mutation (per kilobase) against APOBEC3G mRNA levels (where the level in HEK293 cells is taken as 1) with cell lines sorted according to FeLV restriction phenotype. Results for LCLs, which are discordant by virtue of their low levels of infectious virion release ( Table 2 ) but postinfection accumulation of proviral DNA ( Fig. 3 ), are enclosed by a dashed oval. (C) Percentage of G→A mutations that conform to the A3G signature ( 42 ) for all cell lines in which significant levels of mutations were detected. Blue-gray bars, hematopoietic cells; yellow bars, nonhematopoietic cells. (D) Relative levels of APOBEC3G mRNA (on a log 10 scale, with the level in HEK293 cells taken as 1) for all the cell lines tested, sorted into hematopoietic (blue circles) and nonhematopoietic (green circles) cell lines. Nonpermissive cells (nonspreading, with low virion release) are represented by black circles.
    Power Sybr Green Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8809 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher power sybr green rna to ct rt qpcr kit
    COS hiPSC cohort reprogramming and differentiation. a Validated hiPSCs (from 14 individuals with childhood-onset-schizophrenia (COS) and 12 unrelated healthy controls) and NPCs (12 COS; 12 control individuals) yielded 94 <t>RNA-Seq</t> samples (11 COS; 11 control individuals). b Schematic illustration of the reprogramming and differentiation process, noting the yield at each stage. c Sex breakdown of the COS-control cohort. d Breakdown of SZ-associated copy number variants in the 11 COS patients with RNA-Seq data. e Representative <t>qPCR</t> validation of NANOG, NESTIN , and SYN1 expression in hiPSCs (white bar), NPCs (light gray) and 6-week-old neurons (dark gray) from three individuals. f FACS analysis for pluripotency markers TRA-1-60 (left) and SSEA4 (right) in representative control (blue, n = 17) and COS (red, n = 16) hiPSCs. g FACS analysis for NPC markers SOX2 (left) and NESTIN (right) in control (blue, n = 34) and COS (red, n = 37) NPCs. h Representative images of NPCs (left) and 6-week-old forebrain neurons (right) from control (top) and COS (bottom). NPCs stained with SOX2 (red) and NESTIN (green); neurons stained with MAP2 (red). DAPI-stained nuclei (blue). Scale bar=50 μm. i Computational workflow showing quality control, integration with external data sets, computational deconvolution with Cibersort, decomposition multiple sources of expression variation with variancePartition, coexpression analysis with WGCNA, differential expression and concordance analysis
    Power Sybr Green Rna To Ct Rt Qpcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/power sybr green rna to ct rt qpcr kit/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    power sybr green rna to ct rt qpcr kit - by Bioz Stars, 2020-09
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    Hypermutation of the FeLV-B genome and APOBEC3G mRNA expression in human cells. FeLV genomic sequences (segments of Gag and Pol) were cloned by PCR from infected human cells, and individual templates were sequenced and compared to the reference input virus (pFGB clone). APOBEC3G mRNA expression was determined in the same cells (prior to infection) by quantitative real-time PCR (with SYBR green). (A) Representative plots of hypermutation visualized by the online HYPERMUT program ( www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html ), where sequence changes relative to the reference FeLV-B genome are color coded (red, GG→AG; cyan, GA→AA; green, GC→AC; magenta, GT→AT; black, non-G→A). (B) X/Y plots of G→A mutation (per kilobase) against APOBEC3G mRNA levels (where the level in HEK293 cells is taken as 1) with cell lines sorted according to FeLV restriction phenotype. Results for LCLs, which are discordant by virtue of their low levels of infectious virion release ( Table 2 ) but postinfection accumulation of proviral DNA ( Fig. 3 ), are enclosed by a dashed oval. (C) Percentage of G→A mutations that conform to the A3G signature ( 42 ) for all cell lines in which significant levels of mutations were detected. Blue-gray bars, hematopoietic cells; yellow bars, nonhematopoietic cells. (D) Relative levels of APOBEC3G mRNA (on a log 10 scale, with the level in HEK293 cells taken as 1) for all the cell lines tested, sorted into hematopoietic (blue circles) and nonhematopoietic (green circles) cell lines. Nonpermissive cells (nonspreading, with low virion release) are represented by black circles.

    Journal: Journal of Virology

    Article Title: Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis

    doi: 10.1128/JVI.02119-16

    Figure Lengend Snippet: Hypermutation of the FeLV-B genome and APOBEC3G mRNA expression in human cells. FeLV genomic sequences (segments of Gag and Pol) were cloned by PCR from infected human cells, and individual templates were sequenced and compared to the reference input virus (pFGB clone). APOBEC3G mRNA expression was determined in the same cells (prior to infection) by quantitative real-time PCR (with SYBR green). (A) Representative plots of hypermutation visualized by the online HYPERMUT program ( www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html ), where sequence changes relative to the reference FeLV-B genome are color coded (red, GG→AG; cyan, GA→AA; green, GC→AC; magenta, GT→AT; black, non-G→A). (B) X/Y plots of G→A mutation (per kilobase) against APOBEC3G mRNA levels (where the level in HEK293 cells is taken as 1) with cell lines sorted according to FeLV restriction phenotype. Results for LCLs, which are discordant by virtue of their low levels of infectious virion release ( Table 2 ) but postinfection accumulation of proviral DNA ( Fig. 3 ), are enclosed by a dashed oval. (C) Percentage of G→A mutations that conform to the A3G signature ( 42 ) for all cell lines in which significant levels of mutations were detected. Blue-gray bars, hematopoietic cells; yellow bars, nonhematopoietic cells. (D) Relative levels of APOBEC3G mRNA (on a log 10 scale, with the level in HEK293 cells taken as 1) for all the cell lines tested, sorted into hematopoietic (blue circles) and nonhematopoietic (green circles) cell lines. Nonpermissive cells (nonspreading, with low virion release) are represented by black circles.

    Article Snippet: Aliquots (12.5 ng) of cDNA were amplified in triplicate using Power SYBR green PCR master mix (Thermo Fisher Scientific) and Qiagen QuantiTect primer assays for human hypoxanthine phosphoribosyltransferase (HPRT) (QT00059066), APOBEC3G (QT00070770), SLC20A1 (PIT-1) (QT00028763), or SLC19A2 (THTR1) (QT00007847) on an ABI 7500 real-time PCR system.

    Techniques: Expressing, Genomic Sequencing, Clone Assay, Polymerase Chain Reaction, Infection, Real-time Polymerase Chain Reaction, SYBR Green Assay, Sequencing, Mutagenesis

    The thymus produces a significant number of Pth transcripts that increase after TPTX. (A) The thymus produced Pth transcripts. Mice in which serum PTH levels recovered (n = 10) were euthanized, and the heart, lung, liver, spleen, kidney, pancreas, testis or ovary, hypothalamus, pituitary gland, thymus, and adrenal glands were harvested. RNA qRT-PCR was performed on the tissue samples using SYBR Green/Taqman Master Mix with custom primers for the mouse Pth transcript and a commercial probe-set for 18S rRNA (endogenous control). Expression data were normalized to 18S rRNA, and fold changes of expression against arbitrary numbers were calculated using the 2 − ΔΔ Ct ). (B) Pth transcripts increased after TPTX. Thymi from unoperated mice (control) were harvested, and the same procedures were performed as in (A). qRT-PCR was performed as described with a commercial probe (gray) and custom probes (black). Student t test was performed. ** P

    Journal: Endocrinology

    Article Title: Thymic PTH Increases After Thyroparathyroidectomy in C57BL/KaLwRij Mice

    doi: 10.1210/en.2017-03083

    Figure Lengend Snippet: The thymus produces a significant number of Pth transcripts that increase after TPTX. (A) The thymus produced Pth transcripts. Mice in which serum PTH levels recovered (n = 10) were euthanized, and the heart, lung, liver, spleen, kidney, pancreas, testis or ovary, hypothalamus, pituitary gland, thymus, and adrenal glands were harvested. RNA qRT-PCR was performed on the tissue samples using SYBR Green/Taqman Master Mix with custom primers for the mouse Pth transcript and a commercial probe-set for 18S rRNA (endogenous control). Expression data were normalized to 18S rRNA, and fold changes of expression against arbitrary numbers were calculated using the 2 − ΔΔ Ct ). (B) Pth transcripts increased after TPTX. Thymi from unoperated mice (control) were harvested, and the same procedures were performed as in (A). qRT-PCR was performed as described with a commercial probe (gray) and custom probes (black). Student t test was performed. ** P

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with the Power SYBR Green PCR Master Mix on the Step-One-Plus system (Thermo Fisher Scientific).

    Techniques: Produced, Mouse Assay, Quantitative RT-PCR, SYBR Green Assay, Expressing

    Accumulation of immunostimulatory 5′-ppp-vtRNAs during lytic reactivation. a Predicted secondary structure of vtRNAs generated by RNAfold. b SYBR-Gold staining of in vitro transcribed vtRNAs with or without CIP treatment. c HCT116 ISG54-luciferase reporter cells were transfected with 100 ng in vitro transcribed vtRNAs with or without CIP treatment. Cells were harvested 24 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. d BC-3 cells were reactivated for 3 days and expression of DUSP11 was quantified by RT-qPCR. L latency, D1–D3 lytic reactivation for 1 day to 3 days. The DUSP11 expression was normalized to the level of 18S rRNA and L was set as 1. e Cell lysates were prepared from BC-3 cells described in ( d ) and DUSP11 protein levels were monitored by Western blot. GAPDH was run as a loading control. f Latent and lytic BC-3 cells were subjected to RNAP II ChIP-qPCR analysis. Signals were normalized to input. g ). h HCT116 ISG54-luciferase reporter cells were transfected with vtRNA or U1 RNA isolated by antisense oligonucleotide affinity selection from either latent or lytic BC-3 cells. Cells were harvested 12 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, * p

    Journal: Nature Communications

    Article Title: RIG-I like receptor sensing of host RNAs facilitates the cell-intrinsic immune response to KSHV infection

    doi: 10.1038/s41467-018-07314-7

    Figure Lengend Snippet: Accumulation of immunostimulatory 5′-ppp-vtRNAs during lytic reactivation. a Predicted secondary structure of vtRNAs generated by RNAfold. b SYBR-Gold staining of in vitro transcribed vtRNAs with or without CIP treatment. c HCT116 ISG54-luciferase reporter cells were transfected with 100 ng in vitro transcribed vtRNAs with or without CIP treatment. Cells were harvested 24 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. d BC-3 cells were reactivated for 3 days and expression of DUSP11 was quantified by RT-qPCR. L latency, D1–D3 lytic reactivation for 1 day to 3 days. The DUSP11 expression was normalized to the level of 18S rRNA and L was set as 1. e Cell lysates were prepared from BC-3 cells described in ( d ) and DUSP11 protein levels were monitored by Western blot. GAPDH was run as a loading control. f Latent and lytic BC-3 cells were subjected to RNAP II ChIP-qPCR analysis. Signals were normalized to input. g ). h HCT116 ISG54-luciferase reporter cells were transfected with vtRNA or U1 RNA isolated by antisense oligonucleotide affinity selection from either latent or lytic BC-3 cells. Cells were harvested 12 h posttransfection and subjected to luciferase assay. Mock indicated cells without RNA transfection and was set as 1. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, * p

    Article Snippet: RNA was DNase I (NEB) treated at 37 °C for 20 min, and inactivated with EDTA at 70 °C for 10 min. cDNA was synthesized from DNase-treated RNA with random 9-mer (Integrated DNA Technologies) and M-MLV RT (Promega). qPCR was performed using the PowerUp SYBR Green qPCR kit (Thermo Scientific) with appropriate primers (Supplementary Table ).

    Techniques: Generated, Staining, In Vitro, Luciferase, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Selection

    COS hiPSC cohort reprogramming and differentiation. a Validated hiPSCs (from 14 individuals with childhood-onset-schizophrenia (COS) and 12 unrelated healthy controls) and NPCs (12 COS; 12 control individuals) yielded 94 RNA-Seq samples (11 COS; 11 control individuals). b Schematic illustration of the reprogramming and differentiation process, noting the yield at each stage. c Sex breakdown of the COS-control cohort. d Breakdown of SZ-associated copy number variants in the 11 COS patients with RNA-Seq data. e Representative qPCR validation of NANOG, NESTIN , and SYN1 expression in hiPSCs (white bar), NPCs (light gray) and 6-week-old neurons (dark gray) from three individuals. f FACS analysis for pluripotency markers TRA-1-60 (left) and SSEA4 (right) in representative control (blue, n = 17) and COS (red, n = 16) hiPSCs. g FACS analysis for NPC markers SOX2 (left) and NESTIN (right) in control (blue, n = 34) and COS (red, n = 37) NPCs. h Representative images of NPCs (left) and 6-week-old forebrain neurons (right) from control (top) and COS (bottom). NPCs stained with SOX2 (red) and NESTIN (green); neurons stained with MAP2 (red). DAPI-stained nuclei (blue). Scale bar=50 μm. i Computational workflow showing quality control, integration with external data sets, computational deconvolution with Cibersort, decomposition multiple sources of expression variation with variancePartition, coexpression analysis with WGCNA, differential expression and concordance analysis

    Journal: Nature Communications

    Article Title: Transcriptional signatures of schizophrenia in hiPSC-derived NPCs and neurons are concordant with post-mortem adult brains

    doi: 10.1038/s41467-017-02330-5

    Figure Lengend Snippet: COS hiPSC cohort reprogramming and differentiation. a Validated hiPSCs (from 14 individuals with childhood-onset-schizophrenia (COS) and 12 unrelated healthy controls) and NPCs (12 COS; 12 control individuals) yielded 94 RNA-Seq samples (11 COS; 11 control individuals). b Schematic illustration of the reprogramming and differentiation process, noting the yield at each stage. c Sex breakdown of the COS-control cohort. d Breakdown of SZ-associated copy number variants in the 11 COS patients with RNA-Seq data. e Representative qPCR validation of NANOG, NESTIN , and SYN1 expression in hiPSCs (white bar), NPCs (light gray) and 6-week-old neurons (dark gray) from three individuals. f FACS analysis for pluripotency markers TRA-1-60 (left) and SSEA4 (right) in representative control (blue, n = 17) and COS (red, n = 16) hiPSCs. g FACS analysis for NPC markers SOX2 (left) and NESTIN (right) in control (blue, n = 34) and COS (red, n = 37) NPCs. h Representative images of NPCs (left) and 6-week-old forebrain neurons (right) from control (top) and COS (bottom). NPCs stained with SOX2 (red) and NESTIN (green); neurons stained with MAP2 (red). DAPI-stained nuclei (blue). Scale bar=50 μm. i Computational workflow showing quality control, integration with external data sets, computational deconvolution with Cibersort, decomposition multiple sources of expression variation with variancePartition, coexpression analysis with WGCNA, differential expression and concordance analysis

    Article Snippet: Transcript analysis was carried out using a QuantStudio™ 7 Flex Real-Time PCR System using the Power SYBR green RNA-to-Ct RT-qPCR kit for primers (all ThermoFisher Scientific).

    Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing, FACS, Staining