quantitect taqman probe rt pcr kit  (Qiagen)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    QuantiTect Probe RT PCR Kit
    Description:
    For one step qRT PCR using sequence specific probes for gene expression analysis Kit contents Qiagen QuantiTect Probe RT PCR Kit 200 x 50L rxns RNA Targets Sample One step RT PCR Reaction Sequence specific Probes Ideal for Real time Quantification of RNA Targets For One step qRT PCR Using Sequence specific Probes for Gene Expression Analysis Includes 3 x 1 7mL 2x Qiagen QuantiTect Probe RT PCR Master Mix 100L Qiagen QuantiTect RT Mix 2 x 2mL RNase free Water Benefits Highly sensitive detection of low copy targets Accurate quantification over several logs of template Use of any sequence specific probe on any real time cycler No need to optimize reaction and cycling conditions
    Catalog Number:
    204443
    Price:
    645
    Category:
    QuantiTect Probe RT PCR Kit
    Buy from Supplier


    Structured Review

    Qiagen quantitect taqman probe rt pcr kit
    QuantiTect Probe RT PCR Kit
    For one step qRT PCR using sequence specific probes for gene expression analysis Kit contents Qiagen QuantiTect Probe RT PCR Kit 200 x 50L rxns RNA Targets Sample One step RT PCR Reaction Sequence specific Probes Ideal for Real time Quantification of RNA Targets For One step qRT PCR Using Sequence specific Probes for Gene Expression Analysis Includes 3 x 1 7mL 2x Qiagen QuantiTect Probe RT PCR Master Mix 100L Qiagen QuantiTect RT Mix 2 x 2mL RNase free Water Benefits Highly sensitive detection of low copy targets Accurate quantification over several logs of template Use of any sequence specific probe on any real time cycler No need to optimize reaction and cycling conditions
    https://www.bioz.com/result/quantitect taqman probe rt pcr kit/product/Qiagen
    Average 99 stars, based on 38900 article reviews
    Price from $9.99 to $1999.99
    quantitect taqman probe rt pcr kit - by Bioz Stars, 2021-01
    99/100 stars

    Images

    1) Product Images from "Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis"

    Article Title: Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis

    Journal:

    doi: 10.1099/mic.0.2008/022293-0

    Quantitative TaqMan RT-PCR
    Figure Legend Snippet: Quantitative TaqMan RT-PCR

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    2) Product Images from "The tyrosine kinase inhibitor nilotinib inhibits SARS‐CoV‐2 in vitro. The tyrosine kinase inhibitor nilotinib inhibits SARS‐CoV‐2 in vitro"

    Article Title: The tyrosine kinase inhibitor nilotinib inhibits SARS‐CoV‐2 in vitro. The tyrosine kinase inhibitor nilotinib inhibits SARS‐CoV‐2 in vitro

    Journal: Basic & Clinical Pharmacology & Toxicology

    doi: 10.1111/bcpt.13537

    Inhibitory activity of nilotinib in Vero‐E6 and Calu‐3 cells. Cells were infected with SARS‐CoV‐2 and 1 h post‐inoculation treated with serial dilutions of nilotinib. The infection rate was evaluated at 48 hpi for Vero‐E6 and at 24 hpi for Calu‐3 cells. Results are mean and SEM of 3 independent experiments performed in duplicate
    Figure Legend Snippet: Inhibitory activity of nilotinib in Vero‐E6 and Calu‐3 cells. Cells were infected with SARS‐CoV‐2 and 1 h post‐inoculation treated with serial dilutions of nilotinib. The infection rate was evaluated at 48 hpi for Vero‐E6 and at 24 hpi for Calu‐3 cells. Results are mean and SEM of 3 independent experiments performed in duplicate

    Techniques Used: Activity Assay, Infection

    3) Product Images from "Chronic stress enhances progression of acute lymphoblastic leukemia via ?-adrenergic signaling"

    Article Title: Chronic stress enhances progression of acute lymphoblastic leukemia via ?-adrenergic signaling

    Journal: Brain, Behavior, and Immunity

    doi: 10.1016/j.bbi.2012.01.013

    Effect of chronic restraint stress on ALL progression. (A) 21-day longitudinal growth curves for total body Nalm-6 tumor burden in control and restraint-stressed animals. (B) Nalm-6 tumor burden in separate body areas at Day 14 in control and restraint-stressed
    Figure Legend Snippet: Effect of chronic restraint stress on ALL progression. (A) 21-day longitudinal growth curves for total body Nalm-6 tumor burden in control and restraint-stressed animals. (B) Nalm-6 tumor burden in separate body areas at Day 14 in control and restraint-stressed

    Techniques Used:

    Nalm-6 ALL Model. (A) Nalm-6 pre-B ALL cells were localized and quantified by periodic imaging of leukemia-specific bioluminescence signal from ventral and dorsal sides of SCID mice. (B) Femoral bone marrow white blood cells (WBC) and CD10+ Nalm-6 ALL
    Figure Legend Snippet: Nalm-6 ALL Model. (A) Nalm-6 pre-B ALL cells were localized and quantified by periodic imaging of leukemia-specific bioluminescence signal from ventral and dorsal sides of SCID mice. (B) Femoral bone marrow white blood cells (WBC) and CD10+ Nalm-6 ALL

    Techniques Used: Imaging, Mouse Assay

    β-adrenergic signaling in stress-enhanced ALL progression. (A) Nalm-6 ALL was quantified in control mice vs. stress mice that were treated with or without the β-blocker, propranolol; data representative of 3 independent experiments. (B)
    Figure Legend Snippet: β-adrenergic signaling in stress-enhanced ALL progression. (A) Nalm-6 ALL was quantified in control mice vs. stress mice that were treated with or without the β-blocker, propranolol; data representative of 3 independent experiments. (B)

    Techniques Used: Mouse Assay

    4) Product Images from "Inactivation of the Tulane Virus, a Novel Surrogate for the Human Norovirus"

    Article Title: Inactivation of the Tulane Virus, a Novel Surrogate for the Human Norovirus

    Journal: Journal of food protection

    doi: 10.4315/0362-028X.JFP-12-361

    qRT-PCR
    Figure Legend Snippet: qRT-PCR

    Techniques Used: Quantitative RT-PCR

    5) Product Images from "A novel real‐time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses"

    Article Title: A novel real‐time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses

    Journal: Journal of Medical Virology

    doi: 10.1002/jmv.21962

    Establishment and validation of multivirus real‐time PCR. A : Procedure of the multivirus real‐time PCR system. DNA sample was mixed with Quantitect 2× master mix, and RNA sample was mixed with Quantitect 2× master mix and reverse transcriptase (RT) mix. These mixtures were poured into each well in a 96‐well plate at 10 µl per well. Ten microliters of 2× probe and primer mix were then added to each well in a premixed 96‐well plate. Finally, the virus genes were amplified and detected in a real‐time PCR machine for 2 hr. B : Validation of the multivirus real‐time PCR. A gene expression image by TreeView software based on the results of the multivirus real‐time PCR for control samples is shown. A horizontal line shows each probe–primer set and a vertical line is one sample of positive control. Gray vertical lines indicate no sample. A scale bar indicates copy number of color. A green box indicates specific reactions of target positive controls in specific probe–primer sets. Arrows of (a–c) also show specific signals. The arrow (a) shows positive signal for TTV in a brain sample with both JCV and TTV infection. The arrows (b1–3) show that a probe–primer set for pan‐enterovirus reacted with poliovirus (b1), Coxsackievirus B3 (b2), and Echovirus 6 (b3) positive samples. The arrow (c) shows that a probe–primer set for influenza virus A reacted with H5N1 influenza virus. Details of positive controls were listed in Supplementary Table II.
    Figure Legend Snippet: Establishment and validation of multivirus real‐time PCR. A : Procedure of the multivirus real‐time PCR system. DNA sample was mixed with Quantitect 2× master mix, and RNA sample was mixed with Quantitect 2× master mix and reverse transcriptase (RT) mix. These mixtures were poured into each well in a 96‐well plate at 10 µl per well. Ten microliters of 2× probe and primer mix were then added to each well in a premixed 96‐well plate. Finally, the virus genes were amplified and detected in a real‐time PCR machine for 2 hr. B : Validation of the multivirus real‐time PCR. A gene expression image by TreeView software based on the results of the multivirus real‐time PCR for control samples is shown. A horizontal line shows each probe–primer set and a vertical line is one sample of positive control. Gray vertical lines indicate no sample. A scale bar indicates copy number of color. A green box indicates specific reactions of target positive controls in specific probe–primer sets. Arrows of (a–c) also show specific signals. The arrow (a) shows positive signal for TTV in a brain sample with both JCV and TTV infection. The arrows (b1–3) show that a probe–primer set for pan‐enterovirus reacted with poliovirus (b1), Coxsackievirus B3 (b2), and Echovirus 6 (b3) positive samples. The arrow (c) shows that a probe–primer set for influenza virus A reacted with H5N1 influenza virus. Details of positive controls were listed in Supplementary Table II.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Expressing, Software, Positive Control, Infection

    6) Product Images from "Enhanced pathogenicity of low-pathogenic H9N2 avian influenza virus after vaccination with infectious bronchitis live attenuated vaccine"

    Article Title: Enhanced pathogenicity of low-pathogenic H9N2 avian influenza virus after vaccination with infectious bronchitis live attenuated vaccine

    Journal: Veterinary World

    doi: 10.14202/vetworld.2018.977-985

    Column chart showing RNA copies of H9 using rRT-polymerase chain reaction in chicken groups and different time conditions. (a) Experiment 1, (b) Experiment 2.
    Figure Legend Snippet: Column chart showing RNA copies of H9 using rRT-polymerase chain reaction in chicken groups and different time conditions. (a) Experiment 1, (b) Experiment 2.

    Techniques Used: Polymerase Chain Reaction

    7) Product Images from "β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum"

    Article Title: β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum

    Journal: Brain, behavior, and immunity

    doi: 10.1016/j.bbi.2016.07.162

    Promoter-based bioinformatic analysis of transcription factors in genes differentially-regulated in β-adrenergic-stimulated vs. control macrophages. Data represent mean fold difference (mean ratio of isoproterenol/control) ±SE of transcription
    Figure Legend Snippet: Promoter-based bioinformatic analysis of transcription factors in genes differentially-regulated in β-adrenergic-stimulated vs. control macrophages. Data represent mean fold difference (mean ratio of isoproterenol/control) ±SE of transcription

    Techniques Used:

    Effect of β-adrenergic signaling on select gene transcripts that constitute macrophage activation categories along the M1–M2 spectrum in Murray et al., 2014. Isoproterenol at 1 μM. Data represent mean ± SE of three independent
    Figure Legend Snippet: Effect of β-adrenergic signaling on select gene transcripts that constitute macrophage activation categories along the M1–M2 spectrum in Murray et al., 2014. Isoproterenol at 1 μM. Data represent mean ± SE of three independent

    Techniques Used: Activation Assay

    Mean diagnosticity score for genes that were up-regulated or down-regulated by β-adrenergic signaling. Diagnosticity scores for each gene transcript quantified the extent to which that transcript was predominately expressed by an M2-polarized
    Figure Legend Snippet: Mean diagnosticity score for genes that were up-regulated or down-regulated by β-adrenergic signaling. Diagnosticity scores for each gene transcript quantified the extent to which that transcript was predominately expressed by an M2-polarized

    Techniques Used:

    8) Product Images from "Human retinal ganglion cell axon regeneration by recapitulating developmental mechanisms: effects of recruitment of the mTOR pathway"

    Article Title: Human retinal ganglion cell axon regeneration by recapitulating developmental mechanisms: effects of recruitment of the mTOR pathway

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.178012

    Influence of the mTOR pathway on hRGC differentiation. (A) Schematic of lentivirus-mediated transduction of TSC2 -shRNA in the neural rosette (NR) and their directed differentiation into hRGCs. (B) qPCR analysis of RNA in hRGCs showed a significant decrease in TSC2 transcript levels in hRGCs transduced with TSC2 -shRNA lentivirus versus controls. (C,D) To determine the extent of mTOR signaling in hRGCs transduced with TSC2 -shRNA/control lentivirus or exposed to rapamycin during differentiation, relative levels of p-S6 and S6 were determined by western blot analysis. Levels of p-S6 were increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with controls, whereas those of S6 remained unchanged. (E-H) Immunocytochemical analysis of cells in three treatment groups revealed that the proportion of GFP + (transduced cells) and β-tubulin + cells (nascent RGCs) significantly increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with control. (I-L) The results above were supported by a significant increase and decrease in the proportion of GFP + p-S6 + cells in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, versus controls. (M-P) Similar analysis to quantify S6 + cells revealed an unchanged proportion of GFP + S6 + cells across the groups, demonstrating the specificity of the perturbation of the mTOR pathway. Quantification of immunoreactive cells was performed using 4-5 coverslips/group in 5-6 randomly selected areas. Values are expressed as mean±s.e.m. (Student's t -test). Experiments were carried out three times in triplicate per group for in vitro perturbations. Arrowheads indicate GFP + cells displaying cell type-specific immunoreactivities. Scale bar: 50 µm.
    Figure Legend Snippet: Influence of the mTOR pathway on hRGC differentiation. (A) Schematic of lentivirus-mediated transduction of TSC2 -shRNA in the neural rosette (NR) and their directed differentiation into hRGCs. (B) qPCR analysis of RNA in hRGCs showed a significant decrease in TSC2 transcript levels in hRGCs transduced with TSC2 -shRNA lentivirus versus controls. (C,D) To determine the extent of mTOR signaling in hRGCs transduced with TSC2 -shRNA/control lentivirus or exposed to rapamycin during differentiation, relative levels of p-S6 and S6 were determined by western blot analysis. Levels of p-S6 were increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with controls, whereas those of S6 remained unchanged. (E-H) Immunocytochemical analysis of cells in three treatment groups revealed that the proportion of GFP + (transduced cells) and β-tubulin + cells (nascent RGCs) significantly increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with control. (I-L) The results above were supported by a significant increase and decrease in the proportion of GFP + p-S6 + cells in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, versus controls. (M-P) Similar analysis to quantify S6 + cells revealed an unchanged proportion of GFP + S6 + cells across the groups, demonstrating the specificity of the perturbation of the mTOR pathway. Quantification of immunoreactive cells was performed using 4-5 coverslips/group in 5-6 randomly selected areas. Values are expressed as mean±s.e.m. (Student's t -test). Experiments were carried out three times in triplicate per group for in vitro perturbations. Arrowheads indicate GFP + cells displaying cell type-specific immunoreactivities. Scale bar: 50 µm.

    Techniques Used: Transduction, shRNA, Real-time Polymerase Chain Reaction, Western Blot, In Vitro

    Influence of the mTOR pathway on the expression of regulators of neurites, and axon pathfinding. (A-D) qPCR analysis of RNA in hRGCs in three treatment groups revealed that the expression of transcripts corresponding to positive regulators of axon growth ( KLF6 , SOX11 and GAP43 ) was significantly increased in hRGC TSC2-shRNA groups, compared with controls, but was abrogated in the hRGC Rapamycin group. The patterns of expression of KLF4, a negative regulator of axon growth, was inverse that of KLF6 , SOX11 and GAP43. (E-H) qPCR analysis of RNA in hRGCs in three treatment groups revealed that the expression of transcripts corresponding to axon guidance molecules ( ROBO2 , DCC , EPHB3 and EPHA4 ) significantly increased in hRGC TSC2-shRNA groups, compared with controls, but was abrogated in the hRGC Rapamycin group. Values are expressed as mean±s.e.m. from three independent biological replicates (Student's t -test).
    Figure Legend Snippet: Influence of the mTOR pathway on the expression of regulators of neurites, and axon pathfinding. (A-D) qPCR analysis of RNA in hRGCs in three treatment groups revealed that the expression of transcripts corresponding to positive regulators of axon growth ( KLF6 , SOX11 and GAP43 ) was significantly increased in hRGC TSC2-shRNA groups, compared with controls, but was abrogated in the hRGC Rapamycin group. The patterns of expression of KLF4, a negative regulator of axon growth, was inverse that of KLF6 , SOX11 and GAP43. (E-H) qPCR analysis of RNA in hRGCs in three treatment groups revealed that the expression of transcripts corresponding to axon guidance molecules ( ROBO2 , DCC , EPHB3 and EPHA4 ) significantly increased in hRGC TSC2-shRNA groups, compared with controls, but was abrogated in the hRGC Rapamycin group. Values are expressed as mean±s.e.m. from three independent biological replicates (Student's t -test).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, shRNA, Droplet Countercurrent Chromatography

    9) Product Images from "Hepatitis B Virus DNA is a Substrate for the cGAS/STING Pathway but is not Sensed in Infected Hepatocytes"

    Article Title: Hepatitis B Virus DNA is a Substrate for the cGAS/STING Pathway but is not Sensed in Infected Hepatocytes

    Journal: Viruses

    doi: 10.3390/v12060592

    Hepatocytes respond to foreign DNA. HepG2-hNTCP ( A ), HepG2-hNTCP-control or -cGAS/STING ( B ) or PHH ( C ) were stimulated with cGAMP (A), infected (inf.) with SeV or MVA-gfp, or transfected with HT-DNA or HBV nucleic acids (c/c: copies/cells) ( B , C ). ISG54 mRNA fold inductions to mock ( A , B left part, C ) or to transfection reagent-treated cells (tr. reagent) ( B , right part) was determined by RT-qPCR. Average and SEM of 4 ( A ), 2 ( B ) independent experiments in triplicates or of 4 donors in duplicates ( C ). Levels of significance compared to respective controls ( A , B left part, C : mock; B , right part: transfection reagent-treated cells): **** p
    Figure Legend Snippet: Hepatocytes respond to foreign DNA. HepG2-hNTCP ( A ), HepG2-hNTCP-control or -cGAS/STING ( B ) or PHH ( C ) were stimulated with cGAMP (A), infected (inf.) with SeV or MVA-gfp, or transfected with HT-DNA or HBV nucleic acids (c/c: copies/cells) ( B , C ). ISG54 mRNA fold inductions to mock ( A , B left part, C ) or to transfection reagent-treated cells (tr. reagent) ( B , right part) was determined by RT-qPCR. Average and SEM of 4 ( A ), 2 ( B ) independent experiments in triplicates or of 4 donors in duplicates ( C ). Levels of significance compared to respective controls ( A , B left part, C : mock; B , right part: transfection reagent-treated cells): **** p

    Techniques Used: Infection, Transfection, Quantitative RT-PCR

    HBV DNA is sensed by the cGAS/STING pathway. ( A ) Genome editing of THP1 CRISPR/Cas9 KO for STING, cGAS or MAVS was controlled by Western Blot. ( B ) PMA-differentiated WT or KO THP1 cells were infected with SeV, transfected with HT-DNA (5 pg/cell) or with serial dilutions of undigested (Undig.) or DNAse-digested HBV nucleic acids (n.ac.) from particles. ISG54 mRNA fold induction to the mock was determined by RT-qPCR 24 h post-transfection. Average and SEM of three independent experiments in triplicates are shown. **** p
    Figure Legend Snippet: HBV DNA is sensed by the cGAS/STING pathway. ( A ) Genome editing of THP1 CRISPR/Cas9 KO for STING, cGAS or MAVS was controlled by Western Blot. ( B ) PMA-differentiated WT or KO THP1 cells were infected with SeV, transfected with HT-DNA (5 pg/cell) or with serial dilutions of undigested (Undig.) or DNAse-digested HBV nucleic acids (n.ac.) from particles. ISG54 mRNA fold induction to the mock was determined by RT-qPCR 24 h post-transfection. Average and SEM of three independent experiments in triplicates are shown. **** p

    Techniques Used: CRISPR, Western Blot, Infection, Transfection, Quantitative RT-PCR

    HBV DNAs but not RNAs are immunostimulatory. Total nucleic acids (n.ac.) were extracted from SeV, MVA-gfp or HBV particles. HBV DNA replication intermediates (HBV R.I.) were extracted from HepAD38 cytoplasm. Total RNAs were extracted from HepAD38 or HepG2 cells. When indicated, the nucleic acids were digested with RNAse (blue) or DNAse (red; black bars: undigested). Human MDDCs were transfected with 10-fold dilutions of the nucleic acids (quantified in Table 3 ). ISG54 mRNA level was quantified by RT-qPCR 6 h post-transfection. Average and SEM of three technical replicates of at least two donors are shown. Levels of significance over mock transfection: **** p
    Figure Legend Snippet: HBV DNAs but not RNAs are immunostimulatory. Total nucleic acids (n.ac.) were extracted from SeV, MVA-gfp or HBV particles. HBV DNA replication intermediates (HBV R.I.) were extracted from HepAD38 cytoplasm. Total RNAs were extracted from HepAD38 or HepG2 cells. When indicated, the nucleic acids were digested with RNAse (blue) or DNAse (red; black bars: undigested). Human MDDCs were transfected with 10-fold dilutions of the nucleic acids (quantified in Table 3 ). ISG54 mRNA level was quantified by RT-qPCR 6 h post-transfection. Average and SEM of three technical replicates of at least two donors are shown. Levels of significance over mock transfection: **** p

    Techniques Used: Transfection, Quantitative RT-PCR

    HBVwt or HBV X- infection do not activate nucleic acid-sensing pathways in hepatocytes. HepG2-hNTCP ( A ) or PHH ( B , C ) were infected with HBVwt (MOI 100), HBV X- (MOI 100) or SeV (MOI 0,13). HBV, ISG54 or IFN-λ1 RNAs were quantified by RT-qPCR ( A , B ). Relative expression levels to the reference gene RPL13A were calculated. For ISG54, fold induction to the respective mock-control is shown for each time point. Average and standard error of the mean (SEM) of three independent (HepG2-hNTCP) or 1 (PHH) experiment in technical triplicates are shown. ( C ) Phospho- or total IRF3 protein levels were analyzed by Western blot in HBV-infected PHH (HBVwt or HBV X-, MOI 100).
    Figure Legend Snippet: HBVwt or HBV X- infection do not activate nucleic acid-sensing pathways in hepatocytes. HepG2-hNTCP ( A ) or PHH ( B , C ) were infected with HBVwt (MOI 100), HBV X- (MOI 100) or SeV (MOI 0,13). HBV, ISG54 or IFN-λ1 RNAs were quantified by RT-qPCR ( A , B ). Relative expression levels to the reference gene RPL13A were calculated. For ISG54, fold induction to the respective mock-control is shown for each time point. Average and standard error of the mean (SEM) of three independent (HepG2-hNTCP) or 1 (PHH) experiment in technical triplicates are shown. ( C ) Phospho- or total IRF3 protein levels were analyzed by Western blot in HBV-infected PHH (HBVwt or HBV X-, MOI 100).

    Techniques Used: Infection, Quantitative RT-PCR, Expressing, Western Blot

    10) Product Images from "β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum"

    Article Title: β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum

    Journal: Brain, behavior, and immunity

    doi: 10.1016/j.bbi.2016.07.162

    Effect of β-adrenergic signaling on select gene transcripts that constitute macrophage activation categories along the M1–M2 spectrum in Murray et al., 2014. Isoproterenol at 1 μM. Data represent mean ± SE of three independent
    Figure Legend Snippet: Effect of β-adrenergic signaling on select gene transcripts that constitute macrophage activation categories along the M1–M2 spectrum in Murray et al., 2014. Isoproterenol at 1 μM. Data represent mean ± SE of three independent

    Techniques Used: Activation Assay

    (A) M1 - M2 spectrum categories of macrophage activation, based on the activator and subsequent known transcription factors, as proposed by Peter J. Murray and colleagues following the International Congress of Immunology in Milan in 2013. At the far
    Figure Legend Snippet: (A) M1 - M2 spectrum categories of macrophage activation, based on the activator and subsequent known transcription factors, as proposed by Peter J. Murray and colleagues following the International Congress of Immunology in Milan in 2013. At the far

    Techniques Used: Activation Assay

    11) Product Images from "Targeted Long-Read RNA Sequencing Demonstrates Transcriptional Diversity Driven by Splice-Site Variation in MYBPC3"

    Article Title: Targeted Long-Read RNA Sequencing Demonstrates Transcriptional Diversity Driven by Splice-Site Variation in MYBPC3

    Journal: bioRxiv

    doi: 10.1101/522698

    Study Patient Sample Contains Private Alternatively Spliced Isoforms Targeted sequencing of MYBPC3 cDNA in ten left ventricular heart samples: six healthy heart controls, our MYBPC3 splice-variant study patient, and three HCM samples containing MYH7 point mutations ( MYH7 PM 1-3). Sequencing revealed that our study patient, whose genome contained a splice-site variant in MYBPC3 , contained a highly expressed alternatively spliced isoform. Panel A shows IsoSeq normalized expression (number of full length reads per isoform over the total number of full length reads) of the top 5 highly expressed MYBPC3 isoforms in our study patient identified by PacBio’s IsoSeq pipeline across all ten samples. The canonical isoform is the most highly expressed isoform across all ten samples, but the next two highly expressed isoforms (Alternatively Spliced 1.1 and 1.2, AS1.1 and AS1.2 respectively) are seen only in our study patient sample. SH 1 and SH 2 depict shared alternatively spliced isoforms across both control and disease samples. AS1.1, the next most highly expressed isoform, is depicted in Panel B, and results in the skipping of exon 20. The location of the splice-site variant (located one base prior to the start of exon 20) is denoted with a red triangle.
    Figure Legend Snippet: Study Patient Sample Contains Private Alternatively Spliced Isoforms Targeted sequencing of MYBPC3 cDNA in ten left ventricular heart samples: six healthy heart controls, our MYBPC3 splice-variant study patient, and three HCM samples containing MYH7 point mutations ( MYH7 PM 1-3). Sequencing revealed that our study patient, whose genome contained a splice-site variant in MYBPC3 , contained a highly expressed alternatively spliced isoform. Panel A shows IsoSeq normalized expression (number of full length reads per isoform over the total number of full length reads) of the top 5 highly expressed MYBPC3 isoforms in our study patient identified by PacBio’s IsoSeq pipeline across all ten samples. The canonical isoform is the most highly expressed isoform across all ten samples, but the next two highly expressed isoforms (Alternatively Spliced 1.1 and 1.2, AS1.1 and AS1.2 respectively) are seen only in our study patient sample. SH 1 and SH 2 depict shared alternatively spliced isoforms across both control and disease samples. AS1.1, the next most highly expressed isoform, is depicted in Panel B, and results in the skipping of exon 20. The location of the splice-site variant (located one base prior to the start of exon 20) is denoted with a red triangle.

    Techniques Used: Sequencing, Variant Assay, Expressing

    12) Product Images from "Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer"

    Article Title: Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer

    Journal: Nature Communications

    doi: 10.1038/ncomms4830

    Differential ZAK isoform usage between normal and tumour samples. ( a ) ZAK gene model and protein domain structure. Thick bars: coding exons; thin bars: UTR. TV2 lacks the last nine exons and has a long terminal coding and non-coding exon. Blue and red indicate unique TV1 and TV2 sequences. Transcript variant 1 (TV1) encodes a longer protein product with a sterile alpha motif domain. ( b ) ZAK TV1 fraction is significantly higher in gastric tumours (left) and colon tumours (right), compared with normal adjacent tissues. The fraction of TV1 was measured by the ratio between the number of reads uniquely assignable to TV1 and the number of reads mapped to the entire ZAK gene. To account for smooth muscle contamination in normal tissues, we fit a linear model with smoothelin expression as predictor and the log isoform fraction as response, and used the residuals of the model as the ‘adjusted isoform fraction’. Dots represent samples. Grey lines connect matched tumour and normal samples. The boxes in the box-and-whisker plots represent the interquartile range between the first and third quartiles; the dashed lines (whiskers) extend to the most extreme data points, which is no more than 1.5 times the interquartile range from the box. ( c ) ZAK isoform fractions derived from RNAseq data correlate with quantitative PCR (qPCR) measurements. For nine gastric cancer cell lines in our study, we quantified the ratio between ZAK total expression and ZAK TV1 expression using qPCR, and compared the measurements with the isoform fraction we derived from the RNAseq data. The two measurements have significant correlation (Pearson’s correlation coefficient r =0.91, P -value=0.00077). ( d ) ZAK isoform expression in six TCGA data sets where there are > 10 normal samples. Normal samples are represented by blue dots and tumour samples by red dots. ZAK TV1 fraction is significantly higher (adjusted P -value
    Figure Legend Snippet: Differential ZAK isoform usage between normal and tumour samples. ( a ) ZAK gene model and protein domain structure. Thick bars: coding exons; thin bars: UTR. TV2 lacks the last nine exons and has a long terminal coding and non-coding exon. Blue and red indicate unique TV1 and TV2 sequences. Transcript variant 1 (TV1) encodes a longer protein product with a sterile alpha motif domain. ( b ) ZAK TV1 fraction is significantly higher in gastric tumours (left) and colon tumours (right), compared with normal adjacent tissues. The fraction of TV1 was measured by the ratio between the number of reads uniquely assignable to TV1 and the number of reads mapped to the entire ZAK gene. To account for smooth muscle contamination in normal tissues, we fit a linear model with smoothelin expression as predictor and the log isoform fraction as response, and used the residuals of the model as the ‘adjusted isoform fraction’. Dots represent samples. Grey lines connect matched tumour and normal samples. The boxes in the box-and-whisker plots represent the interquartile range between the first and third quartiles; the dashed lines (whiskers) extend to the most extreme data points, which is no more than 1.5 times the interquartile range from the box. ( c ) ZAK isoform fractions derived from RNAseq data correlate with quantitative PCR (qPCR) measurements. For nine gastric cancer cell lines in our study, we quantified the ratio between ZAK total expression and ZAK TV1 expression using qPCR, and compared the measurements with the isoform fraction we derived from the RNAseq data. The two measurements have significant correlation (Pearson’s correlation coefficient r =0.91, P -value=0.00077). ( d ) ZAK isoform expression in six TCGA data sets where there are > 10 normal samples. Normal samples are represented by blue dots and tumour samples by red dots. ZAK TV1 fraction is significantly higher (adjusted P -value

    Techniques Used: Variant Assay, Expressing, Whisker Assay, Derivative Assay, Real-time Polymerase Chain Reaction

    Experimental validation of ZAK function in cancer. ( a ) Immunoblots of ZAK TV1 and TV2 expressions show that protein level of TV1 is higher in gastric tumours and cell lines, compared with normal stomach tissues. ZAK TV1 was detected with Bethyl α-ZAK antibody and TV2 with Sigma α-ZAK antibody (see Methods). ( b ) ZAK TV1, but not TV2, can stimulate multiple transcriptional programs related to cancer pathways. Transcription reporter assay in 293 cells transfected with empty vector, TV1 or TV2 along with the indicated firefly luciferase reporter construct (AP1, NFkB and TCF/LEF). Activity is normalized to cell number using CellTiter-Glo. Immunoblot shows relative ZAK isoform expression from 293 cells transfected with the indicated construct. ZAK was detected with Sigma α-ZAK antibody. ( c ) Depletion of ZAK from gastric cancer cell lines inhibits cell growth. In cell lines where ZAK knockdown led to reduced viability, there was consistently high TV1 expression, while TV2 expression was marginal and variable (for example, IM-95m cell line, see for example, panel a ). Cell viability analysis was carried out 6 days after infection of gastric cancer cell lines with independent ZAK shRNAs. Cell number is normalized to shNTC-infected cells. Immunoblot indicates the level of ZAK-TV1 depletion 4 days after infection. ZAK was detected with Bethyl α-ZAK antibody.
    Figure Legend Snippet: Experimental validation of ZAK function in cancer. ( a ) Immunoblots of ZAK TV1 and TV2 expressions show that protein level of TV1 is higher in gastric tumours and cell lines, compared with normal stomach tissues. ZAK TV1 was detected with Bethyl α-ZAK antibody and TV2 with Sigma α-ZAK antibody (see Methods). ( b ) ZAK TV1, but not TV2, can stimulate multiple transcriptional programs related to cancer pathways. Transcription reporter assay in 293 cells transfected with empty vector, TV1 or TV2 along with the indicated firefly luciferase reporter construct (AP1, NFkB and TCF/LEF). Activity is normalized to cell number using CellTiter-Glo. Immunoblot shows relative ZAK isoform expression from 293 cells transfected with the indicated construct. ZAK was detected with Sigma α-ZAK antibody. ( c ) Depletion of ZAK from gastric cancer cell lines inhibits cell growth. In cell lines where ZAK knockdown led to reduced viability, there was consistently high TV1 expression, while TV2 expression was marginal and variable (for example, IM-95m cell line, see for example, panel a ). Cell viability analysis was carried out 6 days after infection of gastric cancer cell lines with independent ZAK shRNAs. Cell number is normalized to shNTC-infected cells. Immunoblot indicates the level of ZAK-TV1 depletion 4 days after infection. ZAK was detected with Bethyl α-ZAK antibody.

    Techniques Used: Western Blot, Reporter Assay, Transfection, Plasmid Preparation, Luciferase, Construct, Activity Assay, Expressing, Infection

    13) Product Images from "Evaluation of the kinetics of anti‐ NP and anti‐ HA antibody after infection of Pekin ducks with low pathogenic avian influenza virus"

    Article Title: Evaluation of the kinetics of anti‐ NP and anti‐ HA antibody after infection of Pekin ducks with low pathogenic avian influenza virus

    Journal: Veterinary Medicine and Science

    doi: 10.1002/vms3.18

    Viral excretion of experimentally inoculated ducks. The M viral RNA was detected by RRT ‐ PCR . Results are expressed as the M RNA copy number per ml. Vertical bars represent the results of individual inoculated Pekin ducks. Graphs represent tracheal
    Figure Legend Snippet: Viral excretion of experimentally inoculated ducks. The M viral RNA was detected by RRT ‐ PCR . Results are expressed as the M RNA copy number per ml. Vertical bars represent the results of individual inoculated Pekin ducks. Graphs represent tracheal

    Techniques Used: Quantitative RT-PCR

    14) Product Images from "The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death ▿The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death ▿ ‡"

    Article Title: The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death ▿The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death ▿ ‡

    Journal: Journal of Virology

    doi: 10.1128/JVI.01662-09

    SARS-CoV 3a-GFP partially colocalizes with the Golgi marker Gal-CFP. (A) Vero cells were cotransfected with 3a-GFP and Gal-CFP and analyzed at 24 hpt by confocal microscopy. 3a-GFP (A) and Gal-CFP (B) can be seen to partially colocalize in the merged
    Figure Legend Snippet: SARS-CoV 3a-GFP partially colocalizes with the Golgi marker Gal-CFP. (A) Vero cells were cotransfected with 3a-GFP and Gal-CFP and analyzed at 24 hpt by confocal microscopy. 3a-GFP (A) and Gal-CFP (B) can be seen to partially colocalize in the merged

    Techniques Used: Marker, Confocal Microscopy

    Expression of SARS-CoV 3a is sufficient to cause cell death, vesicle formation, and Golgi fragmentation. (A) Vero cells transfected with 3a-GFP were examined by confocal microscopy 48 hpt after propidium iodide (PI) staining to identify dead cells. (B)
    Figure Legend Snippet: Expression of SARS-CoV 3a is sufficient to cause cell death, vesicle formation, and Golgi fragmentation. (A) Vero cells transfected with 3a-GFP were examined by confocal microscopy 48 hpt after propidium iodide (PI) staining to identify dead cells. (B)

    Techniques Used: Expressing, Transfection, Confocal Microscopy, Staining

    SARS-CoV-infected cells exhibit cytoplamsic vesicles. (A and B) Vero cells were infected for 48 h with WT SARS-CoV and were fixed and analyzed by TEM. Infected cells show numerous vesicles and vacuoles and do not show characteristics of apoptosis such
    Figure Legend Snippet: SARS-CoV-infected cells exhibit cytoplamsic vesicles. (A and B) Vero cells were infected for 48 h with WT SARS-CoV and were fixed and analyzed by TEM. Infected cells show numerous vesicles and vacuoles and do not show characteristics of apoptosis such

    Techniques Used: Infection, Transmission Electron Microscopy

    Cell death caused by SARS-CoV is reduced by deletion of 3a. (A) Vero cells were mock infected (MOCK) or infected with wild-type (WT) or 3a-deficient (Δ3a) SARS-CoV. After 48 h, cells were examined by phase-contrast light microscopy. (B) Quantification
    Figure Legend Snippet: Cell death caused by SARS-CoV is reduced by deletion of 3a. (A) Vero cells were mock infected (MOCK) or infected with wild-type (WT) or 3a-deficient (Δ3a) SARS-CoV. After 48 h, cells were examined by phase-contrast light microscopy. (B) Quantification

    Techniques Used: Infection, Light Microscopy

    Membrane rearrangement and vesicle formation by SARS-CoV is dependent on 3a. (A) Vero cells were infected with WT SARS-CoV and 24 hpi were fixed and analyzed by TEM. Boxed region contains paranuclear vesicles. N, nucleus. (B) TEM of Vero cells either
    Figure Legend Snippet: Membrane rearrangement and vesicle formation by SARS-CoV is dependent on 3a. (A) Vero cells were infected with WT SARS-CoV and 24 hpi were fixed and analyzed by TEM. Boxed region contains paranuclear vesicles. N, nucleus. (B) TEM of Vero cells either

    Techniques Used: Infection, Transmission Electron Microscopy

    Golgi fragmentation caused by SARS-CoV infection depends on 3a. (A) Vero cells were transfected with Golgi marker Gal-CFP and after 24 h were mock infected (MOCK) or infected with SARS-CoV (WT) or Δ3a SARS-CoV (Δ3a) at a multiplicity of
    Figure Legend Snippet: Golgi fragmentation caused by SARS-CoV infection depends on 3a. (A) Vero cells were transfected with Golgi marker Gal-CFP and after 24 h were mock infected (MOCK) or infected with SARS-CoV (WT) or Δ3a SARS-CoV (Δ3a) at a multiplicity of

    Techniques Used: Infection, Transfection, Marker

    15) Product Images from "Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis"

    Article Title: Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-122

    Pharmacodynamic effects of TGF-β antagonists in vivo . A . Protein extracts from snap-frozen uninvolved lung tissue of mice treated with vehicle and LY2109761 and of mice treated with controls and 1D11 were prepared and subject to Western blotting using rabbit phosphoSmad2 antibody (1:1000 dilution). Whole cell lysate from SCP2TR was used as a control (Co). Treatment with both antagonists resulted in a reduction of phosphorylated Smad2 levels. Values represent the means and SD of three mice per group. Unpaired 2-sided t-test was used for comparisons. B . RNA was extracted from snap-frozen uninvolved kidneys of mice treated with vehicle or LY2109761 using Trizol reagent (Invitrogen) and purified using RNeasy mini columns (Qiagen) according to the manufacturer's instructions. Transcript levels of CTGF and PAI-1 were assayed using the QuantiTect™ Probe RT-PCR Kit on a Mx4000 ® Multiplex Quantitative PCR System (Stratagene). Treatment with LY2109761 significantly reduced mRNA levels of both CTGF and PAI-1 relative to GAPDH mRNA. Values represent the means and SD of three mice per group. Unpaired 2-sided t-test was used for comparisons. C . RNA was extracted from snap-frozen lungs of mice treated with vehicle, isotype control antibody or 1D11 and transcript levels determined as described above. No significant reduction in CTGF or PAI-1 mRNA levels could be detected in the 1D11-treated group. Values represent the means and SD of two independent experiments, 3 mice per group. Unpaired 2-sided t-test was used for comparisons.
    Figure Legend Snippet: Pharmacodynamic effects of TGF-β antagonists in vivo . A . Protein extracts from snap-frozen uninvolved lung tissue of mice treated with vehicle and LY2109761 and of mice treated with controls and 1D11 were prepared and subject to Western blotting using rabbit phosphoSmad2 antibody (1:1000 dilution). Whole cell lysate from SCP2TR was used as a control (Co). Treatment with both antagonists resulted in a reduction of phosphorylated Smad2 levels. Values represent the means and SD of three mice per group. Unpaired 2-sided t-test was used for comparisons. B . RNA was extracted from snap-frozen uninvolved kidneys of mice treated with vehicle or LY2109761 using Trizol reagent (Invitrogen) and purified using RNeasy mini columns (Qiagen) according to the manufacturer's instructions. Transcript levels of CTGF and PAI-1 were assayed using the QuantiTect™ Probe RT-PCR Kit on a Mx4000 ® Multiplex Quantitative PCR System (Stratagene). Treatment with LY2109761 significantly reduced mRNA levels of both CTGF and PAI-1 relative to GAPDH mRNA. Values represent the means and SD of three mice per group. Unpaired 2-sided t-test was used for comparisons. C . RNA was extracted from snap-frozen lungs of mice treated with vehicle, isotype control antibody or 1D11 and transcript levels determined as described above. No significant reduction in CTGF or PAI-1 mRNA levels could be detected in the 1D11-treated group. Values represent the means and SD of two independent experiments, 3 mice per group. Unpaired 2-sided t-test was used for comparisons.

    Techniques Used: In Vivo, Mouse Assay, Western Blot, Purification, Reverse Transcription Polymerase Chain Reaction, Multiplex Assay, Real-time Polymerase Chain Reaction

    16) Product Images from "Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients"

    Article Title: Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients

    Journal: mBio

    doi: 10.1128/mBio.00081-15

    Effects of HA mutations on viral replication in primary human airway cells. Small airway epithelial cells were infected with viruses at an MOI of 0.1. The culture supernatants were harvested at 24 (A and D), 48 (B and E), and 72 (C and F) h postinfection and assayed by quantitative real-time RT-PCR to determine the amount of progeny virus RNA. The amounts of progeny virus RNA produced by the HA mutant viruses (A, B, and C) and the seasonal human viruses (D, E, and F) are expressed relative to the amount produced by EG/D1 (wt) virus. Each data point is the mean ± SD from triplicate experiments. Colors on the x axis highlight the categories of HA mutations (A, B, and C) and virus strains (D, E, and F) as indicated. *, P
    Figure Legend Snippet: Effects of HA mutations on viral replication in primary human airway cells. Small airway epithelial cells were infected with viruses at an MOI of 0.1. The culture supernatants were harvested at 24 (A and D), 48 (B and E), and 72 (C and F) h postinfection and assayed by quantitative real-time RT-PCR to determine the amount of progeny virus RNA. The amounts of progeny virus RNA produced by the HA mutant viruses (A, B, and C) and the seasonal human viruses (D, E, and F) are expressed relative to the amount produced by EG/D1 (wt) virus. Each data point is the mean ± SD from triplicate experiments. Colors on the x axis highlight the categories of HA mutations (A, B, and C) and virus strains (D, E, and F) as indicated. *, P

    Techniques Used: Infection, Quantitative RT-PCR, Produced, Mutagenesis

    17) Product Images from "HBV DNA is a substrate for the cGAS/STING pathway but is not sensed in infected hepatocytes"

    Article Title: HBV DNA is a substrate for the cGAS/STING pathway but is not sensed in infected hepatocytes

    Journal: bioRxiv

    doi: 10.1101/867440

    HBV DNA but not RNA is immunostimulatory Total nucleic acids (n.ac.) were extracted from SeV, MVA-gfp or HBV particles. Total RNAs were extracted from HepAD38 or HepG2 cells. When indicated, the nucleic acids were digested with RNase (dark grey) or DNase (light grey; black bars: undigested). Human MDDCs were transfected with 10-fold dilutions of the nucleic acids (quantified in Table 1 ). ISG54 mRNA level was quantified by RT-qPCR 6h post-transfection. Average and SEM of 3 technical replicates of at least 2 donors are shown. Levels of significance: ****p
    Figure Legend Snippet: HBV DNA but not RNA is immunostimulatory Total nucleic acids (n.ac.) were extracted from SeV, MVA-gfp or HBV particles. Total RNAs were extracted from HepAD38 or HepG2 cells. When indicated, the nucleic acids were digested with RNase (dark grey) or DNase (light grey; black bars: undigested). Human MDDCs were transfected with 10-fold dilutions of the nucleic acids (quantified in Table 1 ). ISG54 mRNA level was quantified by RT-qPCR 6h post-transfection. Average and SEM of 3 technical replicates of at least 2 donors are shown. Levels of significance: ****p

    Techniques Used: Transfection, Quantitative RT-PCR

    HBV rcDNA is sensed by the cGAS/STING pathway (A) Genome editing of THP1 CRISPR/Cas9 KO for STING, cGAS or MAVS was controlled by Western Blot. (B) PMA-differentiated WT or KO THP1 cells were infected with SeV, transfected with HT-DNA (0,1 µg/well) or with undigested (Undig.) or DNAse-digested HBV nucleic acids (n.ac.) from particles. ISG54 mRNA fold induction to the mock was determined by RT-qPCR 24h post-transfection. Average and SEM of 3 independent experiments in triplicates are shown. ****p
    Figure Legend Snippet: HBV rcDNA is sensed by the cGAS/STING pathway (A) Genome editing of THP1 CRISPR/Cas9 KO for STING, cGAS or MAVS was controlled by Western Blot. (B) PMA-differentiated WT or KO THP1 cells were infected with SeV, transfected with HT-DNA (0,1 µg/well) or with undigested (Undig.) or DNAse-digested HBV nucleic acids (n.ac.) from particles. ISG54 mRNA fold induction to the mock was determined by RT-qPCR 24h post-transfection. Average and SEM of 3 independent experiments in triplicates are shown. ****p

    Techniques Used: CRISPR, Western Blot, Infection, Transfection, Quantitative RT-PCR

    Hepatocytes respond to foreign DNA HepG2-hNTCP (A), HepG2-hNTCP-control or -cGAS/STING (B) or PHH (C) were stimulated with cGAMP (A), infected (inf.) with SeV or MVA-gfp, or transfected with HT-DNA or HBV nucleic acids (c/c: copies/cells) (B, C). ISG54 mRNA fold inductions to mock (A, B left part, C) or to transfection reagent-treated cells (tr. reagent) (B, right part) was determined by RT-qPCR. Average and SEM of 4 (A), 2 (B) independent experiments in triplicates or of 4 donors in duplicates (C). ****p
    Figure Legend Snippet: Hepatocytes respond to foreign DNA HepG2-hNTCP (A), HepG2-hNTCP-control or -cGAS/STING (B) or PHH (C) were stimulated with cGAMP (A), infected (inf.) with SeV or MVA-gfp, or transfected with HT-DNA or HBV nucleic acids (c/c: copies/cells) (B, C). ISG54 mRNA fold inductions to mock (A, B left part, C) or to transfection reagent-treated cells (tr. reagent) (B, right part) was determined by RT-qPCR. Average and SEM of 4 (A), 2 (B) independent experiments in triplicates or of 4 donors in duplicates (C). ****p

    Techniques Used: Infection, Transfection, Quantitative RT-PCR

    HBV does not affect the expression level of cGAS or STING PHH were infected with HBVwt (MOI 5000). (A) Immunofluorescence staining of HBcAg (red) in mock- or HBV-infected PHH 7 days post infection. cGAS (B) or STING (C) mRNA levels were quantified by RT-qPCR at the indicated times post infection. The HBV entry inhibitor Myrcludex B (MyrB; 1µM) was added as a control. The graph represents the average and standard error of the mean (SEM) of 3 technical replicates of at least 2 donors.
    Figure Legend Snippet: HBV does not affect the expression level of cGAS or STING PHH were infected with HBVwt (MOI 5000). (A) Immunofluorescence staining of HBcAg (red) in mock- or HBV-infected PHH 7 days post infection. cGAS (B) or STING (C) mRNA levels were quantified by RT-qPCR at the indicated times post infection. The HBV entry inhibitor Myrcludex B (MyrB; 1µM) was added as a control. The graph represents the average and standard error of the mean (SEM) of 3 technical replicates of at least 2 donors.

    Techniques Used: Expressing, Infection, Immunofluorescence, Staining, Quantitative RT-PCR

    HBV does not induce an innate response in hepatocytes HepG2-hNTCP (A) or PHH (B, C) were infected with HBVwt (MOI 100) or SeV (MOI 0,13). HBV, ISG54 or IFN-λ1 RNAs were quantified by RT-qPCR (A, B). Relative expression levels to the reference gene RPL13A were calculated. For ISG54, fold induction to the respective mock-control is shown for each time point. Average and standard error of the mean (SEM) of 3 independent (HepG2-hNTCP) or 1 (PHH) experiments in technical triplicates are shown. (C) Phospho- or total IRF3 protein levels were analyzed by Western blot in HBVwt-infected PHH (MOI 100).
    Figure Legend Snippet: HBV does not induce an innate response in hepatocytes HepG2-hNTCP (A) or PHH (B, C) were infected with HBVwt (MOI 100) or SeV (MOI 0,13). HBV, ISG54 or IFN-λ1 RNAs were quantified by RT-qPCR (A, B). Relative expression levels to the reference gene RPL13A were calculated. For ISG54, fold induction to the respective mock-control is shown for each time point. Average and standard error of the mean (SEM) of 3 independent (HepG2-hNTCP) or 1 (PHH) experiments in technical triplicates are shown. (C) Phospho- or total IRF3 protein levels were analyzed by Western blot in HBVwt-infected PHH (MOI 100).

    Techniques Used: Infection, Quantitative RT-PCR, Expressing, Western Blot

    No innate immune response to HBV infection in the absence of HBx HepG2-hNTCP (A) or PHH (B, C) were infected with HBV X- (MOI 100) or with SeV (MOI 0,13). HBV, ISG54 or IFN lambda-1 RNAs were measured by RT-qPCR (A, B). Relative expression levels to the reference gene RPL13A were calculated. For ISG54, fold induction to the respective mock-control is shown for each time point. Average and SEM of 3 independent experiments in triplicates (HepG2-hNTCP) or 1 representative experiment in triplicate (PHH) are shown. (C) Phospho- or total IRF3 protein levels were analyzed by Western blot in infected PHH.
    Figure Legend Snippet: No innate immune response to HBV infection in the absence of HBx HepG2-hNTCP (A) or PHH (B, C) were infected with HBV X- (MOI 100) or with SeV (MOI 0,13). HBV, ISG54 or IFN lambda-1 RNAs were measured by RT-qPCR (A, B). Relative expression levels to the reference gene RPL13A were calculated. For ISG54, fold induction to the respective mock-control is shown for each time point. Average and SEM of 3 independent experiments in triplicates (HepG2-hNTCP) or 1 representative experiment in triplicate (PHH) are shown. (C) Phospho- or total IRF3 protein levels were analyzed by Western blot in infected PHH.

    Techniques Used: Infection, Quantitative RT-PCR, Expressing, Western Blot

    HBV rcDNA as well as replication intermediates are immunostimulatory MDDCs were transfected with 5000 copies/cell and 10-fold serial dilutions of total nucleic acids (n.ac.) from HBV particles or of HBV DNA replication intermediates (HBV R.I.) from HepAD38 cytoplasm. When indicated, the nucleic acids were DNAse-digested (light grey) prior transfection (using the same volume as for 5000 copies/cell of undigested nucleic acids). ISG54 mRNA was quantified by RT-qPCR 6h post-transfection. Average and SEM of 3 technical replicates of at least 2 donors are shown. ****p
    Figure Legend Snippet: HBV rcDNA as well as replication intermediates are immunostimulatory MDDCs were transfected with 5000 copies/cell and 10-fold serial dilutions of total nucleic acids (n.ac.) from HBV particles or of HBV DNA replication intermediates (HBV R.I.) from HepAD38 cytoplasm. When indicated, the nucleic acids were DNAse-digested (light grey) prior transfection (using the same volume as for 5000 copies/cell of undigested nucleic acids). ISG54 mRNA was quantified by RT-qPCR 6h post-transfection. Average and SEM of 3 technical replicates of at least 2 donors are shown. ****p

    Techniques Used: Transfection, Quantitative RT-PCR

    18) Product Images from "Evaluation of the kinetics of anti‐ NP and anti‐ HA antibody after infection of Pekin ducks with low pathogenic avian influenza virus"

    Article Title: Evaluation of the kinetics of anti‐ NP and anti‐ HA antibody after infection of Pekin ducks with low pathogenic avian influenza virus

    Journal: Veterinary Medicine and Science

    doi: 10.1002/vms3.18

    Viral excretion of experimentally inoculated ducks. The M viral RNA was detected by RRT ‐ PCR . Results are expressed as the M RNA copy number per ml. Vertical bars represent the results of individual inoculated Pekin ducks. Graphs represent tracheal or cloacal excretion of virus by ducks inoculated with H7N1/chicken virus (a, b), H3N1/domestic duck virus (c, d) or H7N7/Canada goose virus (e, f). No swabs (tracheal or cloacal) were collected at 1 dpi for H3N1/domestic duck virus‐inoculated ducks.
    Figure Legend Snippet: Viral excretion of experimentally inoculated ducks. The M viral RNA was detected by RRT ‐ PCR . Results are expressed as the M RNA copy number per ml. Vertical bars represent the results of individual inoculated Pekin ducks. Graphs represent tracheal or cloacal excretion of virus by ducks inoculated with H7N1/chicken virus (a, b), H3N1/domestic duck virus (c, d) or H7N7/Canada goose virus (e, f). No swabs (tracheal or cloacal) were collected at 1 dpi for H3N1/domestic duck virus‐inoculated ducks.

    Techniques Used: Quantitative RT-PCR

    Related Articles

    Multiplex Assay:

    Article Title: A novel real‐time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses
    Article Snippet: .. In addition, the duplex real‐time PCR using Quantitect Multiplex Probe RT‐PCR kit (Qiagen) had similar sensitivity to single real‐time PCR procedures using Quantitect Probe RT‐PCR kit (Qiagen) in several probe–primer sets (Supplementary Fig. 1B). .. Gene Expression Image A gene expression image was produced with TreeView and Cluster software by Michael Eisen, University of California at Berkeley ( http://rana.lbl.gov/EisenSoftware.htm ) [Eisen et al., ].

    Amplification:

    Article Title: Evaluation of the kinetics of anti‐ NP and anti‐ HA antibody after infection of Pekin ducks with low pathogenic avian influenza virus
    Article Snippet: .. The Quantitect Probe RRT‐PCR kit (QiagenGmBH, Hilden, Germany) was used for amplification using a Biosystems 7500 Real‐Time PCR Cycler (Applied Biosystems, Lennik, Belgium). ..

    Infection:

    Article Title: Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients
    Article Snippet: .. Progeny virus RNA was extracted from the culture medium of infected cells with a QIAamp viral RNA minikit (Qiagen) and assayed by quantitative real-time reverse transcription (RT)-PCR (QuantiTect probe RT-PCR kit, Qiagen), using primers targeting the M gene as previously described ( ). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Evaluation of the kinetics of anti‐ NP and anti‐ HA antibody after infection of Pekin ducks with low pathogenic avian influenza virus
    Article Snippet: .. The Quantitect Probe RRT‐PCR kit (QiagenGmBH, Hilden, Germany) was used for amplification using a Biosystems 7500 Real‐Time PCR Cycler (Applied Biosystems, Lennik, Belgium). ..

    Article Title: A novel real‐time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses
    Article Snippet: .. In addition, the duplex real‐time PCR using Quantitect Multiplex Probe RT‐PCR kit (Qiagen) had similar sensitivity to single real‐time PCR procedures using Quantitect Probe RT‐PCR kit (Qiagen) in several probe–primer sets (Supplementary Fig. 1B). .. Gene Expression Image A gene expression image was produced with TreeView and Cluster software by Michael Eisen, University of California at Berkeley ( http://rana.lbl.gov/EisenSoftware.htm ) [Eisen et al., ].

    Article Title: Inactivation of the Tulane Virus, a Novel Surrogate for the Human Norovirus
    Article Snippet: .. Extracted viral RNA was quantitated with probe-based qRT-PCR by using a qPCR system (MX3000P, Stratagene, La Jolla, CA) with a one-step qRT-PCR kit (QuantiTect Probe RT-PCR Kit, QIAGEN) in accordance with the manufacturer’s protocol. .. The P289-P290 amplicon of TV in the RNA-dependent RNA polymerase gene of caliciviruses ( , ) was determined in silico with DNASTAR software (Madison, WI) and experimentally confirmed (results not shown here).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis
    Article Snippet: .. Real-time quantitative RT-PCR Transcript levels of individual genes were assayed in frozen tissue specimens by quantitative real time (qRT)-PCR, using the QuantiTect™ Probe RT-PCR Kit (QIAGEN, Valencia, CA). .. For the PCR, 50 μl reactions were set up with 100 ng of RNA, 0.4 μM primer, 0.2 μM dual labeled probe, 0.5 μl of QuantiTect™ Reverse Transcriptase Mix and QuantiTect™ Probe RT-PCR Master Mix.

    Article Title: A Novel Squirrel Respirovirus with Putative Zoonotic Potential
    Article Snippet: .. Cycling conditions followed the QuantiTect Probe RT-PCR Kit (Qiagen) manufacturer protocol. .. For validation, RNA covering the amplification site of MRV and HRV1 was transcribed in vitro using T7-Polymerase (Thermo Fisher) as described by the manufacturer, followed by DNase digestion (TURBO DNA-free™ Kit, Thermo Fisher), and spiked into background squirrel RNA.

    Article Title: Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis
    Article Snippet: .. Real-time RT-PCR was used to determine the quantitative levels of SAP1–10 mRNA transcripts in RNA samples using the GeneAmp ABI 5700 and 7500 PCR machines (PE Applied Biosystems) and the QuantiTect TaqMan probe RT-PCR kit (Qiagen) according to the manufacturer’s instructions. .. Each reaction mixture consisted of 1× RT-PCR buffer, 4 mM MgCl2 , 600 nM forward and reverse primer, 200 nM TaqMan probe, HotStarTaq DNA polymerase, omniscript and sensiscript RT enzymes, and template RNA.

    Article Title: A novel real‐time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses
    Article Snippet: .. In addition, the duplex real‐time PCR using Quantitect Multiplex Probe RT‐PCR kit (Qiagen) had similar sensitivity to single real‐time PCR procedures using Quantitect Probe RT‐PCR kit (Qiagen) in several probe–primer sets (Supplementary Fig. 1B). .. Gene Expression Image A gene expression image was produced with TreeView and Cluster software by Michael Eisen, University of California at Berkeley ( http://rana.lbl.gov/EisenSoftware.htm ) [Eisen et al., ].

    Article Title: Inactivation of the Tulane Virus, a Novel Surrogate for the Human Norovirus
    Article Snippet: .. Extracted viral RNA was quantitated with probe-based qRT-PCR by using a qPCR system (MX3000P, Stratagene, La Jolla, CA) with a one-step qRT-PCR kit (QuantiTect Probe RT-PCR Kit, QIAGEN) in accordance with the manufacturer’s protocol. .. The P289-P290 amplicon of TV in the RNA-dependent RNA polymerase gene of caliciviruses ( , ) was determined in silico with DNASTAR software (Madison, WI) and experimentally confirmed (results not shown here).

    Article Title: Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients
    Article Snippet: .. Progeny virus RNA was extracted from the culture medium of infected cells with a QIAamp viral RNA minikit (Qiagen) and assayed by quantitative real-time reverse transcription (RT)-PCR (QuantiTect probe RT-PCR kit, Qiagen), using primers targeting the M gene as previously described ( ). ..

    Quantitative RT-PCR:

    Article Title: Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis
    Article Snippet: .. Real-time quantitative RT-PCR Transcript levels of individual genes were assayed in frozen tissue specimens by quantitative real time (qRT)-PCR, using the QuantiTect™ Probe RT-PCR Kit (QIAGEN, Valencia, CA). .. For the PCR, 50 μl reactions were set up with 100 ng of RNA, 0.4 μM primer, 0.2 μM dual labeled probe, 0.5 μl of QuantiTect™ Reverse Transcriptase Mix and QuantiTect™ Probe RT-PCR Master Mix.

    Article Title: Evaluation of the kinetics of anti‐ NP and anti‐ HA antibody after infection of Pekin ducks with low pathogenic avian influenza virus
    Article Snippet: .. The Quantitect Probe RRT‐PCR kit (QiagenGmBH, Hilden, Germany) was used for amplification using a Biosystems 7500 Real‐Time PCR Cycler (Applied Biosystems, Lennik, Belgium). ..

    Article Title: Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis
    Article Snippet: .. Real-time RT-PCR was used to determine the quantitative levels of SAP1–10 mRNA transcripts in RNA samples using the GeneAmp ABI 5700 and 7500 PCR machines (PE Applied Biosystems) and the QuantiTect TaqMan probe RT-PCR kit (Qiagen) according to the manufacturer’s instructions. .. Each reaction mixture consisted of 1× RT-PCR buffer, 4 mM MgCl2 , 600 nM forward and reverse primer, 200 nM TaqMan probe, HotStarTaq DNA polymerase, omniscript and sensiscript RT enzymes, and template RNA.

    Article Title: Inactivation of the Tulane Virus, a Novel Surrogate for the Human Norovirus
    Article Snippet: .. Extracted viral RNA was quantitated with probe-based qRT-PCR by using a qPCR system (MX3000P, Stratagene, La Jolla, CA) with a one-step qRT-PCR kit (QuantiTect Probe RT-PCR Kit, QIAGEN) in accordance with the manufacturer’s protocol. .. The P289-P290 amplicon of TV in the RNA-dependent RNA polymerase gene of caliciviruses ( , ) was determined in silico with DNASTAR software (Madison, WI) and experimentally confirmed (results not shown here).

    Polymerase Chain Reaction:

    Article Title: Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis
    Article Snippet: .. Real-time RT-PCR was used to determine the quantitative levels of SAP1–10 mRNA transcripts in RNA samples using the GeneAmp ABI 5700 and 7500 PCR machines (PE Applied Biosystems) and the QuantiTect TaqMan probe RT-PCR kit (Qiagen) according to the manufacturer’s instructions. .. Each reaction mixture consisted of 1× RT-PCR buffer, 4 mM MgCl2 , 600 nM forward and reverse primer, 200 nM TaqMan probe, HotStarTaq DNA polymerase, omniscript and sensiscript RT enzymes, and template RNA.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Qiagen quantitect taqman probe rt pcr kit
    Quantitative <t>TaqMan</t> <t>RT-PCR</t>
    Quantitect Taqman Probe Rt Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitect taqman probe rt pcr kit/product/Qiagen
    Average 99 stars, based on 200 article reviews
    Price from $9.99 to $1999.99
    quantitect taqman probe rt pcr kit - by Bioz Stars, 2021-01
    99/100 stars
      Buy from Supplier

    92
    Qiagen superscript iii rt kit
    Influence of the mTOR pathway on hRGC differentiation. (A) Schematic of lentivirus-mediated transduction of TSC2 -shRNA in the neural rosette (NR) and their directed differentiation into hRGCs. (B) qPCR analysis of <t>RNA</t> in hRGCs showed a significant decrease in TSC2 transcript levels in hRGCs transduced with TSC2 -shRNA lentivirus versus controls. (C,D) To determine the extent of mTOR signaling in hRGCs transduced with TSC2 -shRNA/control lentivirus or exposed to rapamycin during differentiation, relative levels of p-S6 and S6 were determined by western blot analysis. Levels of p-S6 were increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with controls, whereas those of S6 remained unchanged. (E-H) Immunocytochemical analysis of cells in <t>three</t> treatment groups revealed that the proportion of GFP + (transduced cells) and β-tubulin + cells (nascent RGCs) significantly increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with control. (I-L) The results above were supported by a significant increase and decrease in the proportion of GFP + p-S6 + cells in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, versus controls. (M-P) Similar analysis to quantify S6 + cells revealed an unchanged proportion of GFP + S6 + cells across the groups, demonstrating the specificity of the perturbation of the mTOR pathway. Quantification of immunoreactive cells was performed using 4-5 coverslips/group in 5-6 randomly selected areas. Values are expressed as mean±s.e.m. (Student's t -test). Experiments were carried out three times in triplicate per group for in vitro perturbations. Arrowheads indicate GFP + cells displaying cell type-specific immunoreactivities. Scale bar: 50 µm.
    Superscript Iii Rt Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii rt kit/product/Qiagen
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    superscript iii rt kit - by Bioz Stars, 2021-01
    92/100 stars
      Buy from Supplier

    Image Search Results


    Quantitative TaqMan RT-PCR

    Journal:

    Article Title: Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis

    doi: 10.1099/mic.0.2008/022293-0

    Figure Lengend Snippet: Quantitative TaqMan RT-PCR

    Article Snippet: Real-time RT-PCR was used to determine the quantitative levels of SAP1–10 mRNA transcripts in RNA samples using the GeneAmp ABI 5700 and 7500 PCR machines (PE Applied Biosystems) and the QuantiTect TaqMan probe RT-PCR kit (Qiagen) according to the manufacturer’s instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    qRT-PCR

    Journal: Journal of food protection

    Article Title: Inactivation of the Tulane Virus, a Novel Surrogate for the Human Norovirus

    doi: 10.4315/0362-028X.JFP-12-361

    Figure Lengend Snippet: qRT-PCR

    Article Snippet: Extracted viral RNA was quantitated with probe-based qRT-PCR by using a qPCR system (MX3000P, Stratagene, La Jolla, CA) with a one-step qRT-PCR kit (QuantiTect Probe RT-PCR Kit, QIAGEN) in accordance with the manufacturer’s protocol.

    Techniques: Quantitative RT-PCR

    Establishment and validation of multivirus real‐time PCR. A : Procedure of the multivirus real‐time PCR system. DNA sample was mixed with Quantitect 2× master mix, and RNA sample was mixed with Quantitect 2× master mix and reverse transcriptase (RT) mix. These mixtures were poured into each well in a 96‐well plate at 10 µl per well. Ten microliters of 2× probe and primer mix were then added to each well in a premixed 96‐well plate. Finally, the virus genes were amplified and detected in a real‐time PCR machine for 2 hr. B : Validation of the multivirus real‐time PCR. A gene expression image by TreeView software based on the results of the multivirus real‐time PCR for control samples is shown. A horizontal line shows each probe–primer set and a vertical line is one sample of positive control. Gray vertical lines indicate no sample. A scale bar indicates copy number of color. A green box indicates specific reactions of target positive controls in specific probe–primer sets. Arrows of (a–c) also show specific signals. The arrow (a) shows positive signal for TTV in a brain sample with both JCV and TTV infection. The arrows (b1–3) show that a probe–primer set for pan‐enterovirus reacted with poliovirus (b1), Coxsackievirus B3 (b2), and Echovirus 6 (b3) positive samples. The arrow (c) shows that a probe–primer set for influenza virus A reacted with H5N1 influenza virus. Details of positive controls were listed in Supplementary Table II.

    Journal: Journal of Medical Virology

    Article Title: A novel real‐time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses

    doi: 10.1002/jmv.21962

    Figure Lengend Snippet: Establishment and validation of multivirus real‐time PCR. A : Procedure of the multivirus real‐time PCR system. DNA sample was mixed with Quantitect 2× master mix, and RNA sample was mixed with Quantitect 2× master mix and reverse transcriptase (RT) mix. These mixtures were poured into each well in a 96‐well plate at 10 µl per well. Ten microliters of 2× probe and primer mix were then added to each well in a premixed 96‐well plate. Finally, the virus genes were amplified and detected in a real‐time PCR machine for 2 hr. B : Validation of the multivirus real‐time PCR. A gene expression image by TreeView software based on the results of the multivirus real‐time PCR for control samples is shown. A horizontal line shows each probe–primer set and a vertical line is one sample of positive control. Gray vertical lines indicate no sample. A scale bar indicates copy number of color. A green box indicates specific reactions of target positive controls in specific probe–primer sets. Arrows of (a–c) also show specific signals. The arrow (a) shows positive signal for TTV in a brain sample with both JCV and TTV infection. The arrows (b1–3) show that a probe–primer set for pan‐enterovirus reacted with poliovirus (b1), Coxsackievirus B3 (b2), and Echovirus 6 (b3) positive samples. The arrow (c) shows that a probe–primer set for influenza virus A reacted with H5N1 influenza virus. Details of positive controls were listed in Supplementary Table II.

    Article Snippet: In addition, the duplex real‐time PCR using Quantitect Multiplex Probe RT‐PCR kit (Qiagen) had similar sensitivity to single real‐time PCR procedures using Quantitect Probe RT‐PCR kit (Qiagen) in several probe–primer sets (Supplementary Fig. 1B).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Expressing, Software, Positive Control, Infection

    Influence of the mTOR pathway on hRGC differentiation. (A) Schematic of lentivirus-mediated transduction of TSC2 -shRNA in the neural rosette (NR) and their directed differentiation into hRGCs. (B) qPCR analysis of RNA in hRGCs showed a significant decrease in TSC2 transcript levels in hRGCs transduced with TSC2 -shRNA lentivirus versus controls. (C,D) To determine the extent of mTOR signaling in hRGCs transduced with TSC2 -shRNA/control lentivirus or exposed to rapamycin during differentiation, relative levels of p-S6 and S6 were determined by western blot analysis. Levels of p-S6 were increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with controls, whereas those of S6 remained unchanged. (E-H) Immunocytochemical analysis of cells in three treatment groups revealed that the proportion of GFP + (transduced cells) and β-tubulin + cells (nascent RGCs) significantly increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with control. (I-L) The results above were supported by a significant increase and decrease in the proportion of GFP + p-S6 + cells in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, versus controls. (M-P) Similar analysis to quantify S6 + cells revealed an unchanged proportion of GFP + S6 + cells across the groups, demonstrating the specificity of the perturbation of the mTOR pathway. Quantification of immunoreactive cells was performed using 4-5 coverslips/group in 5-6 randomly selected areas. Values are expressed as mean±s.e.m. (Student's t -test). Experiments were carried out three times in triplicate per group for in vitro perturbations. Arrowheads indicate GFP + cells displaying cell type-specific immunoreactivities. Scale bar: 50 µm.

    Journal: Development (Cambridge, England)

    Article Title: Human retinal ganglion cell axon regeneration by recapitulating developmental mechanisms: effects of recruitment of the mTOR pathway

    doi: 10.1242/dev.178012

    Figure Lengend Snippet: Influence of the mTOR pathway on hRGC differentiation. (A) Schematic of lentivirus-mediated transduction of TSC2 -shRNA in the neural rosette (NR) and their directed differentiation into hRGCs. (B) qPCR analysis of RNA in hRGCs showed a significant decrease in TSC2 transcript levels in hRGCs transduced with TSC2 -shRNA lentivirus versus controls. (C,D) To determine the extent of mTOR signaling in hRGCs transduced with TSC2 -shRNA/control lentivirus or exposed to rapamycin during differentiation, relative levels of p-S6 and S6 were determined by western blot analysis. Levels of p-S6 were increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with controls, whereas those of S6 remained unchanged. (E-H) Immunocytochemical analysis of cells in three treatment groups revealed that the proportion of GFP + (transduced cells) and β-tubulin + cells (nascent RGCs) significantly increased and decreased in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, compared with control. (I-L) The results above were supported by a significant increase and decrease in the proportion of GFP + p-S6 + cells in hRGC TSC2-shRNA and hRGC Rapamycin groups, respectively, versus controls. (M-P) Similar analysis to quantify S6 + cells revealed an unchanged proportion of GFP + S6 + cells across the groups, demonstrating the specificity of the perturbation of the mTOR pathway. Quantification of immunoreactive cells was performed using 4-5 coverslips/group in 5-6 randomly selected areas. Values are expressed as mean±s.e.m. (Student's t -test). Experiments were carried out three times in triplicate per group for in vitro perturbations. Arrowheads indicate GFP + cells displaying cell type-specific immunoreactivities. Scale bar: 50 µm.

    Article Snippet: Five μg of total RNA per sample was used for reverse transcription into cDNA, using Superscript III RT kit, following the manufacturer's instructions.

    Techniques: Transduction, shRNA, Real-time Polymerase Chain Reaction, Western Blot, In Vitro

    Influence of the mTOR pathway on the expression of regulators of neurites, and axon pathfinding. (A-D) qPCR analysis of RNA in hRGCs in three treatment groups revealed that the expression of transcripts corresponding to positive regulators of axon growth ( KLF6 , SOX11 and GAP43 ) was significantly increased in hRGC TSC2-shRNA groups, compared with controls, but was abrogated in the hRGC Rapamycin group. The patterns of expression of KLF4, a negative regulator of axon growth, was inverse that of KLF6 , SOX11 and GAP43. (E-H) qPCR analysis of RNA in hRGCs in three treatment groups revealed that the expression of transcripts corresponding to axon guidance molecules ( ROBO2 , DCC , EPHB3 and EPHA4 ) significantly increased in hRGC TSC2-shRNA groups, compared with controls, but was abrogated in the hRGC Rapamycin group. Values are expressed as mean±s.e.m. from three independent biological replicates (Student's t -test).

    Journal: Development (Cambridge, England)

    Article Title: Human retinal ganglion cell axon regeneration by recapitulating developmental mechanisms: effects of recruitment of the mTOR pathway

    doi: 10.1242/dev.178012

    Figure Lengend Snippet: Influence of the mTOR pathway on the expression of regulators of neurites, and axon pathfinding. (A-D) qPCR analysis of RNA in hRGCs in three treatment groups revealed that the expression of transcripts corresponding to positive regulators of axon growth ( KLF6 , SOX11 and GAP43 ) was significantly increased in hRGC TSC2-shRNA groups, compared with controls, but was abrogated in the hRGC Rapamycin group. The patterns of expression of KLF4, a negative regulator of axon growth, was inverse that of KLF6 , SOX11 and GAP43. (E-H) qPCR analysis of RNA in hRGCs in three treatment groups revealed that the expression of transcripts corresponding to axon guidance molecules ( ROBO2 , DCC , EPHB3 and EPHA4 ) significantly increased in hRGC TSC2-shRNA groups, compared with controls, but was abrogated in the hRGC Rapamycin group. Values are expressed as mean±s.e.m. from three independent biological replicates (Student's t -test).

    Article Snippet: Five μg of total RNA per sample was used for reverse transcription into cDNA, using Superscript III RT kit, following the manufacturer's instructions.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, shRNA, Droplet Countercurrent Chromatography