taqman universal pcr master mix  (Thermo Fisher)


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    TaqMan Universal PCR Master Mix
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    Alternative Product Try TaqMan Fast Advanced Master Mix our highest performance probe based master mix With TaqMan Fast Advanced Master Mix we ve taken the best of TaqMan Universal PCR Master Mix and added additional capabilities for your gene expression analysis Applied Biosystems TaqMan Universal PCR Master Mix is the ideal reagent solution when you need a Master Mix for multiple 5 nuclease DNA applications Applied Biosystems reagents have been validated with TaqMan Assays and Applied Biosystems real time systems to ensure sensitive accurate and reliable performance every time • Contains AmpliTaq Gold DNA polymerase to provide a better yield and a more robust 5 nuclease assay than AmpliTaq DNA polymerase• Includes a passive internal reference based on proprietary ROX dye for superb precision on Applied Biosystems real time PCR instruments • Buffer enhancements guarantee performance and reliability in all applications even with G C rich sequences• AmpErase UNG protects against subsequent re amplification from PCR products containing dU to minimize carry over contamination• Rapid assay development guidelines are provided to minimize optimization time
    Catalog Number:
    4304437
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    Kits and Assays
    Applications:
    Enzymes & Master Mixes for Real-Time PCR|Industrial & Applied Science|PCR & Real-Time PCR|PCR-Based Drug Discovery Assays|Pharma & Biopharma|RNAi, Epigenetics & Non-Coding RNA Research|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Real Time PCR-Based miRNA Analysis|miRNA & Non-Coding RNA Analysis|Target & Lead Identification & Validation|Gene Expression Analysis & Genotyping
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    Structured Review

    Thermo Fisher taqman universal pcr master mix
    MiRNA validation. Nineteen miRNAs from our deep sequencing data were selected for further validation using real-time <t>TaqMan®</t> MicroRNA Assays, including nine novel, cat-specific miRNAs. miR-151 and miR-361 were expressed at very consistent levels in all of the deep sequencing samples (not shown) and, thus, <t>qRT-PCR</t> values were normalized to these two miRs. Figure shows heat maps of averaged and normalized miR counts from the deep sequencing data and of the normalized relative expression from qRT-PCR. All of the normalized deep sequencing and qRT-PCR values for each individual miR in each individual sample are given in Supplementary Table S1.10 .
    Alternative Product Try TaqMan Fast Advanced Master Mix our highest performance probe based master mix With TaqMan Fast Advanced Master Mix we ve taken the best of TaqMan Universal PCR Master Mix and added additional capabilities for your gene expression analysis Applied Biosystems TaqMan Universal PCR Master Mix is the ideal reagent solution when you need a Master Mix for multiple 5 nuclease DNA applications Applied Biosystems reagents have been validated with TaqMan Assays and Applied Biosystems real time systems to ensure sensitive accurate and reliable performance every time • Contains AmpliTaq Gold DNA polymerase to provide a better yield and a more robust 5 nuclease assay than AmpliTaq DNA polymerase• Includes a passive internal reference based on proprietary ROX dye for superb precision on Applied Biosystems real time PCR instruments • Buffer enhancements guarantee performance and reliability in all applications even with G C rich sequences• AmpErase UNG protects against subsequent re amplification from PCR products containing dU to minimize carry over contamination• Rapid assay development guidelines are provided to minimize optimization time
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    1) Product Images from "Discovery and characterization of the feline miRNAome"

    Article Title: Discovery and characterization of the feline miRNAome

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10164-w

    MiRNA validation. Nineteen miRNAs from our deep sequencing data were selected for further validation using real-time TaqMan® MicroRNA Assays, including nine novel, cat-specific miRNAs. miR-151 and miR-361 were expressed at very consistent levels in all of the deep sequencing samples (not shown) and, thus, qRT-PCR values were normalized to these two miRs. Figure shows heat maps of averaged and normalized miR counts from the deep sequencing data and of the normalized relative expression from qRT-PCR. All of the normalized deep sequencing and qRT-PCR values for each individual miR in each individual sample are given in Supplementary Table S1.10 .
    Figure Legend Snippet: MiRNA validation. Nineteen miRNAs from our deep sequencing data were selected for further validation using real-time TaqMan® MicroRNA Assays, including nine novel, cat-specific miRNAs. miR-151 and miR-361 were expressed at very consistent levels in all of the deep sequencing samples (not shown) and, thus, qRT-PCR values were normalized to these two miRs. Figure shows heat maps of averaged and normalized miR counts from the deep sequencing data and of the normalized relative expression from qRT-PCR. All of the normalized deep sequencing and qRT-PCR values for each individual miR in each individual sample are given in Supplementary Table S1.10 .

    Techniques Used: Sequencing, Quantitative RT-PCR, Expressing

    2) Product Images from "Paradoxical effect of lenalidomide on cytokine/growth factor profiles in multiple myeloma"

    Article Title: Paradoxical effect of lenalidomide on cytokine/growth factor profiles in multiple myeloma

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2013.186

    Lenalidomide modulates cytokines and IGF-1 profiles in myeloma cells. ( A ) Expression of TNF- α mRNA in myeloma cells and BMSC. ( B ) Modulation of TNF- α mRNA induced by LEN treatment in myeloma cells and BMSC. Human myeloma cell lines, primary CD138+ MM cells and BMSCs were incubated with or without 10 μ ℳ LEN for 24 h. Total RNA was extracted and reverse-transcribed as described previously. Tumour necrosis factor- α mRNA levels were evaluated by real-time PCR using the TaqMan probe Hs00174128-m1 (Applied Biosystem). The relative expression of TNF- α mRNA was calculated according to the equation of Pfaffl and normalised to LP1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate. ( C ) Increase of TNF- α production by LEN in myeloma cell lines. ( D ) Expression of IGF-1 mRNA in myeloma cells and BMSCs. ( E ) Increase in IGF-1 mRNA levels under LEN treatment. Insulin-like growth factor-1 mRNA levels were evaluated as above using the TaqMan gene expression assay Hs 00153126-m1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate.
    Figure Legend Snippet: Lenalidomide modulates cytokines and IGF-1 profiles in myeloma cells. ( A ) Expression of TNF- α mRNA in myeloma cells and BMSC. ( B ) Modulation of TNF- α mRNA induced by LEN treatment in myeloma cells and BMSC. Human myeloma cell lines, primary CD138+ MM cells and BMSCs were incubated with or without 10 μ ℳ LEN for 24 h. Total RNA was extracted and reverse-transcribed as described previously. Tumour necrosis factor- α mRNA levels were evaluated by real-time PCR using the TaqMan probe Hs00174128-m1 (Applied Biosystem). The relative expression of TNF- α mRNA was calculated according to the equation of Pfaffl and normalised to LP1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate. ( C ) Increase of TNF- α production by LEN in myeloma cell lines. ( D ) Expression of IGF-1 mRNA in myeloma cells and BMSCs. ( E ) Increase in IGF-1 mRNA levels under LEN treatment. Insulin-like growth factor-1 mRNA levels were evaluated as above using the TaqMan gene expression assay Hs 00153126-m1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate.

    Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction

    3) Product Images from "Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair"

    Article Title: Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.02.016

    hPSC-Derived MesoT Displays Molecular Characteristics of Primary Mesothelium (A) Sources of cells used for RNA-seq analysis. hPSC-derived MesoT cells and tdTomato+ mesothelium isolated from mouse embryonic gut, liver, heart, and lungs (embryonic day 15.5 [E15.5]) were compared to RNA-seq data in the public domain. (B) Addition of Wnt3a, BMP4, and retinoic acid (RA) to SplM (ISL1 + , NKX2.5 + , ZO1 + ) efficiently generates MesoT (WT1 + , αSMA + , VIM + ) with a mesenchymal phenotype. Scale bars, 50 μm. (C) qRT-PCR data showing fold-change of transcript levels for markers of SplM ( ISL1, NKX2.5, and GATA4 ) and MesoT ( WT1, TBX18, and TCF21 ) following directed differentiation of human embryonic stem cells (hESCs, WA09). TaqMan assays for each transcript were performed in technical triplicate and fold-change shown relative to untreated hESCs (WA09) after normalization with 18S RNA. .
    Figure Legend Snippet: hPSC-Derived MesoT Displays Molecular Characteristics of Primary Mesothelium (A) Sources of cells used for RNA-seq analysis. hPSC-derived MesoT cells and tdTomato+ mesothelium isolated from mouse embryonic gut, liver, heart, and lungs (embryonic day 15.5 [E15.5]) were compared to RNA-seq data in the public domain. (B) Addition of Wnt3a, BMP4, and retinoic acid (RA) to SplM (ISL1 + , NKX2.5 + , ZO1 + ) efficiently generates MesoT (WT1 + , αSMA + , VIM + ) with a mesenchymal phenotype. Scale bars, 50 μm. (C) qRT-PCR data showing fold-change of transcript levels for markers of SplM ( ISL1, NKX2.5, and GATA4 ) and MesoT ( WT1, TBX18, and TCF21 ) following directed differentiation of human embryonic stem cells (hESCs, WA09). TaqMan assays for each transcript were performed in technical triplicate and fold-change shown relative to untreated hESCs (WA09) after normalization with 18S RNA. .

    Techniques Used: Derivative Assay, RNA Sequencing Assay, Isolation, Quantitative RT-PCR

    4) Product Images from "A Hox gene mutation that triggers nonsense-mediated RNA decay and affects alternative splicing during Drosophila development"

    Article Title: A Hox gene mutation that triggers nonsense-mediated RNA decay and affects alternative splicing during Drosophila development

    Journal: Nucleic Acids Research

    doi:

    Quantification of Ubx isoforms in wild type and Ubx 195 embryos. ( A ) The location of primers (arrows) and Taqman probes (rectangles) is indicated. Please note that Taqman probes only hybridise to isoform-specific exon–exon junctions. This experimental design also prevents the detection of non-specific signals from traces of genomic DNA present in RNA preparations. ( B ) Amplification plots of real-time PCRs using Ubx Ia , Ubx IVa and Rp49 probes on wild type and Ubx 195 templates (late embryos). The plots relate PCR cycle number to changes (Δ) in detected fluorescence with background removed (Rn) on a logarithmic scale. Arrows highlight threshold cycle values (Ct) obtained for each genotype in the different reactions. The lower the Ct values, the higher the abundance of the RNA species detected by the system.
    Figure Legend Snippet: Quantification of Ubx isoforms in wild type and Ubx 195 embryos. ( A ) The location of primers (arrows) and Taqman probes (rectangles) is indicated. Please note that Taqman probes only hybridise to isoform-specific exon–exon junctions. This experimental design also prevents the detection of non-specific signals from traces of genomic DNA present in RNA preparations. ( B ) Amplification plots of real-time PCRs using Ubx Ia , Ubx IVa and Rp49 probes on wild type and Ubx 195 templates (late embryos). The plots relate PCR cycle number to changes (Δ) in detected fluorescence with background removed (Rn) on a logarithmic scale. Arrows highlight threshold cycle values (Ct) obtained for each genotype in the different reactions. The lower the Ct values, the higher the abundance of the RNA species detected by the system.

    Techniques Used: Amplification, IA, Polymerase Chain Reaction, Fluorescence

    5) Product Images from "Dual AAV Gene Therapy for Duchenne Muscular Dystrophy with a 7-kb Mini-Dystrophin Gene in the Canine Model"

    Article Title: Dual AAV Gene Therapy for Duchenne Muscular Dystrophy with a 7-kb Mini-Dystrophin Gene in the Canine Model

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2017.095

    AAV genome copy quantification revealed accumulation of dual AAV vectors in the injected muscle. TaqMan quantitative PCR was performed to measure the AAV genome copy number in the treated and untreated ECU muscles and gastrocnemius muscle in Dog E25. Tissues from an affected dog that did not receive AAV injection were used as the negative control. (A) AAV genome copy number results for YL37. (B) AAV genome copy number results for YL39. ECU, extensor carpi ulnaris.
    Figure Legend Snippet: AAV genome copy quantification revealed accumulation of dual AAV vectors in the injected muscle. TaqMan quantitative PCR was performed to measure the AAV genome copy number in the treated and untreated ECU muscles and gastrocnemius muscle in Dog E25. Tissues from an affected dog that did not receive AAV injection were used as the negative control. (A) AAV genome copy number results for YL37. (B) AAV genome copy number results for YL39. ECU, extensor carpi ulnaris.

    Techniques Used: Injection, Real-time Polymerase Chain Reaction, Negative Control

    6) Product Images from "Pro-atherogenic lung and oral pathogens induce an inflammatory response in human and mouse mast cells"

    Article Title: Pro-atherogenic lung and oral pathogens induce an inflammatory response in human and mouse mast cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2008.00285.x

    Effect of pro-atherogenic bacteria on human mast cell cytokine expression. Cultured human mast cells were infected with Aggregatibacter actinomycetemcomitans (Aa) strains AT445b and JP2 or Chlamydia pneumoniae (Cpn) in a cell-to-bacteria ratio of 1: 10 and incubated for 6 hrs. mRNA expressions for IL-8 ( A ), TNF-α ( B ) and monocyte chemoattractant protein-1 (MCP-1) ( C ) were analysed by real-time PCR using TaqMan chemistry. Data are normalized to β-actin and presented as means ± SD.
    Figure Legend Snippet: Effect of pro-atherogenic bacteria on human mast cell cytokine expression. Cultured human mast cells were infected with Aggregatibacter actinomycetemcomitans (Aa) strains AT445b and JP2 or Chlamydia pneumoniae (Cpn) in a cell-to-bacteria ratio of 1: 10 and incubated for 6 hrs. mRNA expressions for IL-8 ( A ), TNF-α ( B ) and monocyte chemoattractant protein-1 (MCP-1) ( C ) were analysed by real-time PCR using TaqMan chemistry. Data are normalized to β-actin and presented as means ± SD.

    Techniques Used: Expressing, Cell Culture, Infection, Incubation, Real-time Polymerase Chain Reaction

    7) Product Images from "MyomiRs as Markers of Insulin Resistance and Decreased Myogenesis in Skeletal Muscle of Diet-Induced Obese Mice"

    Article Title: MyomiRs as Markers of Insulin Resistance and Decreased Myogenesis in Skeletal Muscle of Diet-Induced Obese Mice

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2016.00076

    Time-course of muscle-specific microRNAs miR-133a (A), miR-1a (B), and miR-206 (E) expression in soleus muscle of high-fat diet (HFD) and control diet (CD)-fed mice . Total RNA was extracted from soleus muscle of HFD- and CD-fed mice, and 10 ng was used for stem-loop reverse transcription. RT products were used for TaqMan real-time PCR. SnoRNA-202 was used for normalization. * p
    Figure Legend Snippet: Time-course of muscle-specific microRNAs miR-133a (A), miR-1a (B), and miR-206 (E) expression in soleus muscle of high-fat diet (HFD) and control diet (CD)-fed mice . Total RNA was extracted from soleus muscle of HFD- and CD-fed mice, and 10 ng was used for stem-loop reverse transcription. RT products were used for TaqMan real-time PCR. SnoRNA-202 was used for normalization. * p

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

    8) Product Images from "Insights into the complex regulation of rpoS in Borrelia burgdorferi"

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2007.05813.x

    Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).
    Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Techniques Used: Quantitative RT-PCR, Standard Deviation

    Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).
    Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Techniques Used: Quantitative RT-PCR, Standard Deviation

    Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.
    Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

    Techniques Used: Quantitative RT-PCR, Standard Deviation

    9) Product Images from "Dysregulated miR-183 inhibits migration in breast cancer cells"

    Article Title: Dysregulated miR-183 inhibits migration in breast cancer cells

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-10-502

    Dysregulation of mRNAs following induced overexpression of miR-183 . Figure 5a : 14 genes were differentially expressed in T47D cells transfected with pre- miR-183 compared to negative control. Gene expression was analysed using TLDA. Figure 5b : RQ-PCR using conventional taqman assays verified downregulation of a selection of breast cancer associated genes following transfection of T47D cells with pre-miR-183.
    Figure Legend Snippet: Dysregulation of mRNAs following induced overexpression of miR-183 . Figure 5a : 14 genes were differentially expressed in T47D cells transfected with pre- miR-183 compared to negative control. Gene expression was analysed using TLDA. Figure 5b : RQ-PCR using conventional taqman assays verified downregulation of a selection of breast cancer associated genes following transfection of T47D cells with pre-miR-183.

    Techniques Used: Over Expression, Transfection, Negative Control, Expressing, TLDA Assay, Polymerase Chain Reaction, Selection

    10) Product Images from "Bromodomain and extra-terminal domain inhibition modulates the expression of pathologically relevant microRNAs in diffuse large B-cell lymphoma"

    Article Title: Bromodomain and extra-terminal domain inhibition modulates the expression of pathologically relevant microRNAs in diffuse large B-cell lymphoma

    Journal: Haematologica

    doi: 10.3324/haematol.2018.191684

    OTX015 modulates microRNA-96-5p expression in diffuse large B-cell lymphoma models. (A) OTX015 upregulates miR-96-5p in a time-dependent manner. Two germinal center B-cell (GCB)-diffuse large B-cell lymphoma (DLBCL) cell lines (DOHH-2, OCI-LY-1) and two activated B-cell (ABC)-DLBCL cell lines (SU-DHL-2, HBL-1) were treated with dimethyl sulfoxide (DMSO) or 500 nM OTX015 for 4, 24, and 48 h. Expression of miR-96-5p was determined by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR). Expression of RNU6B was used for normalization. For each time-point, the mean fold-change relative to the DMSO control is shown. (B) OTX015 treatment of DLBCL cells downregulates PRMT5. Two GCB-DLBCL (DOHH-2, OCI-LY-1) and two ABC-DLBCL (SU-DHL-2, HBL-1) cell lines were treated with DMSO or 500 nM OTX015 for 4, 24, and 48 h. Expression of PRMT5 was determined by qRT-PCR. GAPDH expression was used for normalization. For each time-point, the mean fold-change relative to the DMSO control is shown. (C) OTX015 reduces PRMT5 ) and normalized to GAPDH signals. Representative images of two independent Western blot analyses are shown. The graphs show the mean of three independent experiments. ** P
    Figure Legend Snippet: OTX015 modulates microRNA-96-5p expression in diffuse large B-cell lymphoma models. (A) OTX015 upregulates miR-96-5p in a time-dependent manner. Two germinal center B-cell (GCB)-diffuse large B-cell lymphoma (DLBCL) cell lines (DOHH-2, OCI-LY-1) and two activated B-cell (ABC)-DLBCL cell lines (SU-DHL-2, HBL-1) were treated with dimethyl sulfoxide (DMSO) or 500 nM OTX015 for 4, 24, and 48 h. Expression of miR-96-5p was determined by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR). Expression of RNU6B was used for normalization. For each time-point, the mean fold-change relative to the DMSO control is shown. (B) OTX015 treatment of DLBCL cells downregulates PRMT5. Two GCB-DLBCL (DOHH-2, OCI-LY-1) and two ABC-DLBCL (SU-DHL-2, HBL-1) cell lines were treated with DMSO or 500 nM OTX015 for 4, 24, and 48 h. Expression of PRMT5 was determined by qRT-PCR. GAPDH expression was used for normalization. For each time-point, the mean fold-change relative to the DMSO control is shown. (C) OTX015 reduces PRMT5 ) and normalized to GAPDH signals. Representative images of two independent Western blot analyses are shown. The graphs show the mean of three independent experiments. ** P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    11) Product Images from "Immunodominant fragments of myelin basic protein initiate T cell-dependent pain"

    Article Title: Immunodominant fragments of myelin basic protein initiate T cell-dependent pain

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-9-119

    Acute MMP-9/2 inhibition attenuates CCI-induced allodynia and neuroinflammation. ( A ) Gelatin zymography of sciatic nerve extracts (70 μg total protein/lane) obtained at 3 h, 6 h, 1 day, 3 days, 5 days, and 7 days post-CCI. The arrows point to the MMP-9 and MMP-2 species. ( B ) Taqman qRT-PCR for MMP-9 in the sham (Sh) and CCI sciatic nerves (day 1). The mean relative mRNA ± SEM of n = 6/group normalized to GAPDH compared to naïve (N) nerve (**, P
    Figure Legend Snippet: Acute MMP-9/2 inhibition attenuates CCI-induced allodynia and neuroinflammation. ( A ) Gelatin zymography of sciatic nerve extracts (70 μg total protein/lane) obtained at 3 h, 6 h, 1 day, 3 days, 5 days, and 7 days post-CCI. The arrows point to the MMP-9 and MMP-2 species. ( B ) Taqman qRT-PCR for MMP-9 in the sham (Sh) and CCI sciatic nerves (day 1). The mean relative mRNA ± SEM of n = 6/group normalized to GAPDH compared to naïve (N) nerve (**, P

    Techniques Used: Inhibition, Zymography, Quantitative RT-PCR

    12) Product Images from "High-Fidelity Single Molecule Quantification in a Flow Cytometer Using Multiparametric Optical Analysis"

    Article Title: High-Fidelity Single Molecule Quantification in a Flow Cytometer Using Multiparametric Optical Analysis

    Journal: ACS nano

    doi: 10.1021/acsnano.9b09498

    Detection of fluorescently labeled and extended microRNA in a flow cytometer. (a,b) Scatter plots show events detected for microRNA amplicons labeled with both dye-DNA probes and SYBR Green. (a) Correlation between side scattering and SYBR Green fluorescence intensity. Black lines indicate gates. (b) Same measurement as (a) showing Cy5 and Cy3 intensity channels for each event. (c) Correlation between molecular counts and microRNA concentration with all other conditions held constant. Red line indicates the linear dynamic range. (d) microRNA quantification through reverse transcription polymerase chain reaction. Plot shows the measured cycle threshold for each microRNA concentration using TaqMan qRT-PCR reagents and a real-time PCR instrument. All data points and error bars represent the mean and standard deviation, respectively, of three technical replicates.
    Figure Legend Snippet: Detection of fluorescently labeled and extended microRNA in a flow cytometer. (a,b) Scatter plots show events detected for microRNA amplicons labeled with both dye-DNA probes and SYBR Green. (a) Correlation between side scattering and SYBR Green fluorescence intensity. Black lines indicate gates. (b) Same measurement as (a) showing Cy5 and Cy3 intensity channels for each event. (c) Correlation between molecular counts and microRNA concentration with all other conditions held constant. Red line indicates the linear dynamic range. (d) microRNA quantification through reverse transcription polymerase chain reaction. Plot shows the measured cycle threshold for each microRNA concentration using TaqMan qRT-PCR reagents and a real-time PCR instrument. All data points and error bars represent the mean and standard deviation, respectively, of three technical replicates.

    Techniques Used: Labeling, Flow Cytometry, SYBR Green Assay, Fluorescence, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Standard Deviation

    13) Product Images from "miR-524-5p of the primate-specific C19MC miRNA cluster targets TP53IPN1- and EMT-associated genes to regulate cellular reprogramming"

    Article Title: miR-524-5p of the primate-specific C19MC miRNA cluster targets TP53IPN1- and EMT-associated genes to regulate cellular reprogramming

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0666-3

    miR-524-5p regulates critical features of cellular reprogramming via targeting TP53INP1. a Endogenous transcript levels of miR-524-5p and TP53INP1 in WJ0706 cells. Before (mock) or after transfection of a miR-524-5p mimic, WJ0706 cells were harvested 48 h post-transfection for analysis; miR-524-5p copy number was determined by Taqman qRT-PCR ( left panel ); TP53INP1 transcript level was determined by direct RT-PCR ( right panel ). b – f The miR-524-5p mimic-transfected WJ0706 cells were subjected to assays to determine cell proliferation ( b , c ), cell viability ( d ), apoptosis after oxidative stress induced with 200 μM H 2 O 2 ( e ), and expression of selected pluripotency genes ( f ). All the experiments also included mock transfection with a negative control ( NC ) miRNA mimic, or a TP53INP1 siRNA (siTP53INP1) included as controls. For effects on cell proliferation, cell counts at different days post-transfection ( b ), or by BrdU ELISA measurements ( c ) were performed. For apoptosis assay ( d , e ), cell viability 2 h after incubation with H 2 O 2 was determined by the MTT assay ( d ), or the histone-associated DNA fragments of apoptotic cells were quantified by ELISA assay 6 h after H 2 O 2 treatment ( e ). f Expression of pluripotency genes in the transfected cells was analyzed by real-time RT-PCR 48 h post-transfection. Relative absorbance unit and mRNA level were determined as the relative absorbance units and expression, respectively to the value of the mock and negative control experiments, respectively, which was set as 1.0. * p
    Figure Legend Snippet: miR-524-5p regulates critical features of cellular reprogramming via targeting TP53INP1. a Endogenous transcript levels of miR-524-5p and TP53INP1 in WJ0706 cells. Before (mock) or after transfection of a miR-524-5p mimic, WJ0706 cells were harvested 48 h post-transfection for analysis; miR-524-5p copy number was determined by Taqman qRT-PCR ( left panel ); TP53INP1 transcript level was determined by direct RT-PCR ( right panel ). b – f The miR-524-5p mimic-transfected WJ0706 cells were subjected to assays to determine cell proliferation ( b , c ), cell viability ( d ), apoptosis after oxidative stress induced with 200 μM H 2 O 2 ( e ), and expression of selected pluripotency genes ( f ). All the experiments also included mock transfection with a negative control ( NC ) miRNA mimic, or a TP53INP1 siRNA (siTP53INP1) included as controls. For effects on cell proliferation, cell counts at different days post-transfection ( b ), or by BrdU ELISA measurements ( c ) were performed. For apoptosis assay ( d , e ), cell viability 2 h after incubation with H 2 O 2 was determined by the MTT assay ( d ), or the histone-associated DNA fragments of apoptotic cells were quantified by ELISA assay 6 h after H 2 O 2 treatment ( e ). f Expression of pluripotency genes in the transfected cells was analyzed by real-time RT-PCR 48 h post-transfection. Relative absorbance unit and mRNA level were determined as the relative absorbance units and expression, respectively to the value of the mock and negative control experiments, respectively, which was set as 1.0. * p

    Techniques Used: Transfection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Enzyme-linked Immunosorbent Assay, Apoptosis Assay, Incubation, MTT Assay

    14) Product Images from "Loss of MicroRNA Targets in the 3? Untranslated Region as a Mechanism of Retroviral Insertional Activation of Growth Factor Independence 1 ▿"

    Article Title: Loss of MicroRNA Targets in the 3? Untranslated Region as a Mechanism of Retroviral Insertional Activation of Growth Factor Independence 1 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00427-09

    (A) Gfi1 expression in MLV-induced lymphomas. TaqMan real-time PCR was performed on 43 tumors harboring integrations in the Gfi1 3′UTR and elsewhere in the gfi1 locus as well as on the three control tissues, i.e., thymus (T), spleen (S), and mesenteric
    Figure Legend Snippet: (A) Gfi1 expression in MLV-induced lymphomas. TaqMan real-time PCR was performed on 43 tumors harboring integrations in the Gfi1 3′UTR and elsewhere in the gfi1 locus as well as on the three control tissues, i.e., thymus (T), spleen (S), and mesenteric

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    15) Product Images from "Tailor-made RNAi knockdown against triplet repeat disease-causing alleles"

    Article Title: Tailor-made RNAi knockdown against triplet repeat disease-causing alleles

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1012153107

    Biased recovery of disease-causing HTT alleles. ( A ) cDNAs prepared from two HD cases (C0142 and C0221) were subjected to the pull-down method (+), and then the length of the CAG trinucleotide repeat array in HTT was investigated by PCR analysis. Untreated cDNAs (−) were also examined as a control. ( B ) cDNAs (C0142 and C0221), which were the same as in A , were subjected to the TaqMan SNP Genotyping assay, and the results were plotted in blue. Standard curves (indicated in black) were constructed by the same SNP typing using serially diluted (0.01, 1, 10 ng) untreated cDNAs. The examined coding SNP (cSNP) sites are indicated. x - and y -axes indicate relative fluorescent intensity obtained from the TaqMan probes against the indicated alleles, respectively, and allelic nucleotides at the cSNP sites are also shown in parentheses. ( C ) Linkage between the expanded CAG trinucleotide repeats and cSNPs was estimated in each HD case according to the results of the cSNP typing in B . ( D ) Confirmatory experiments by conventional methods. The same SNP typing as in B was carried out using the isolated plasmids carrying the HTT ). Dots in blue and open circles represent the results of the plasmids containing the mutant and normal HTT .
    Figure Legend Snippet: Biased recovery of disease-causing HTT alleles. ( A ) cDNAs prepared from two HD cases (C0142 and C0221) were subjected to the pull-down method (+), and then the length of the CAG trinucleotide repeat array in HTT was investigated by PCR analysis. Untreated cDNAs (−) were also examined as a control. ( B ) cDNAs (C0142 and C0221), which were the same as in A , were subjected to the TaqMan SNP Genotyping assay, and the results were plotted in blue. Standard curves (indicated in black) were constructed by the same SNP typing using serially diluted (0.01, 1, 10 ng) untreated cDNAs. The examined coding SNP (cSNP) sites are indicated. x - and y -axes indicate relative fluorescent intensity obtained from the TaqMan probes against the indicated alleles, respectively, and allelic nucleotides at the cSNP sites are also shown in parentheses. ( C ) Linkage between the expanded CAG trinucleotide repeats and cSNPs was estimated in each HD case according to the results of the cSNP typing in B . ( D ) Confirmatory experiments by conventional methods. The same SNP typing as in B was carried out using the isolated plasmids carrying the HTT ). Dots in blue and open circles represent the results of the plasmids containing the mutant and normal HTT .

    Techniques Used: Polymerase Chain Reaction, TaqMan SNP Genotyping Assay, Construct, Isolation, Mutagenesis

    16) Product Images from "A SNP in pri-miR-10a is associated with recurrent spontaneous abortion in a Han-Chinese population"

    Article Title: A SNP in pri-miR-10a is associated with recurrent spontaneous abortion in a Han-Chinese population

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7002

    Sequencing, secondary structure prediction and miR-10a expression detection ( A ) An example of chromatographs showing the rs3809783 in pri-miR-10a. Black arrows indicate SNP site. ( B ) Predicted structure of pri-miR-10a. RNA fold web server was used to predict the secondary structure of pri-miR-10a. ( C ) MiR-10a expression analysis. MiR-10a level was detected in cells transfected with the empty pCR3.1 vector, pCR3.1-miR-10a-AA, pCR3.1-miR-10a-TT or pCR3.1-miR-10a-A/T by TaqMan miRNA RT-Real Time PCR. U6 snRNA was used as internal control. The relative level of miR-10a was normalized to U6. ** P
    Figure Legend Snippet: Sequencing, secondary structure prediction and miR-10a expression detection ( A ) An example of chromatographs showing the rs3809783 in pri-miR-10a. Black arrows indicate SNP site. ( B ) Predicted structure of pri-miR-10a. RNA fold web server was used to predict the secondary structure of pri-miR-10a. ( C ) MiR-10a expression analysis. MiR-10a level was detected in cells transfected with the empty pCR3.1 vector, pCR3.1-miR-10a-AA, pCR3.1-miR-10a-TT or pCR3.1-miR-10a-A/T by TaqMan miRNA RT-Real Time PCR. U6 snRNA was used as internal control. The relative level of miR-10a was normalized to U6. ** P

    Techniques Used: Sequencing, Expressing, Transfection, Plasmid Preparation, miRNA RT, Real-time Polymerase Chain Reaction

    17) Product Images from "miR-122 does not impact recognition of the HCV genome by innate sensors of RNA but rather protects the 5′ end from the cellular pyrophosphatases, DOM3Z and DUSP11"

    Article Title: miR-122 does not impact recognition of the HCV genome by innate sensors of RNA but rather protects the 5′ end from the cellular pyrophosphatases, DOM3Z and DUSP11

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky273

    miR-122 binding does not shield the 5′ terminus of HCV RNA against RLR recognition. Huh-7 cells were electroporated with siRIG-I or siControl (siCon) at day –3 and at day 0 cells were electroporated again with the indicated siRNA and either ( A ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA, or ( B ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( C ) Western blot showing knockdown efficiency with antibodies against RIG-I and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. Huh-7 cells were electroporated with siMDA5 or siControl (siCon) at day –3, at day 0 cells were electroporated again with the indicated siRNA, and either ( D ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or ( E ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( F ) Quantitative PCR analysis indicating knockdown efficiency using MDA5-specific and GAPDH-specific TaqMan probes. MDA5 mRNA levels were calculated relative to the siCon. ( G ) Huh-7 cells were electroporated with siMDA5 on day –3, treated with 50 IU/ml IFN-α on day –1 and harvested for western blot at day 0 using antibodies against MDA5 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. ( H ) Northern blot analysis of FL HCV RNA accumulation during MDA5 knockdown at day 3. ( I ) Densitometry quantification of northern blot analysis in (H) normalized to siCon. Huh-7 cells were electroporated with siLGP2 or siControl (siCon) at day –2, at day 0 cells were electroporated again with the indicated siRNA, and either ( J ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or ( K ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( L ) Western blot showing knockdown efficiency with antibodies against LGP2 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. All data are representative of at least three independent experiments and statistical significance was determined by paired parametric t test.
    Figure Legend Snippet: miR-122 binding does not shield the 5′ terminus of HCV RNA against RLR recognition. Huh-7 cells were electroporated with siRIG-I or siControl (siCon) at day –3 and at day 0 cells were electroporated again with the indicated siRNA and either ( A ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA, or ( B ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( C ) Western blot showing knockdown efficiency with antibodies against RIG-I and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. Huh-7 cells were electroporated with siMDA5 or siControl (siCon) at day –3, at day 0 cells were electroporated again with the indicated siRNA, and either ( D ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or ( E ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( F ) Quantitative PCR analysis indicating knockdown efficiency using MDA5-specific and GAPDH-specific TaqMan probes. MDA5 mRNA levels were calculated relative to the siCon. ( G ) Huh-7 cells were electroporated with siMDA5 on day –3, treated with 50 IU/ml IFN-α on day –1 and harvested for western blot at day 0 using antibodies against MDA5 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. ( H ) Northern blot analysis of FL HCV RNA accumulation during MDA5 knockdown at day 3. ( I ) Densitometry quantification of northern blot analysis in (H) normalized to siCon. Huh-7 cells were electroporated with siLGP2 or siControl (siCon) at day –2, at day 0 cells were electroporated again with the indicated siRNA, and either ( J ) wild-type or GNN FL HCV RNA, and a firefly luciferase control mRNA or ( K ) S1+S2p3 SGR or S1+S2p3 GND SGR, a Renilla luciferase control mRNA, and miR-122p3 (miR-122-dependent) or miCon (miR-122-independent). Replication was measured by evaluating luciferase production at the indicated timepoints. ( L ) Western blot showing knockdown efficiency with antibodies against LGP2 and β-actin. Percent knockdown ± standard deviation relative to siCon is indicated. All data are representative of at least three independent experiments and statistical significance was determined by paired parametric t test.

    Techniques Used: Binding Assay, Luciferase, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction, Northern Blot

    18) Product Images from "Differential Expression of microRNAs in Francisella tularensis-Infected Human Macrophages: miR-155-Dependent Downregulation of MyD88 Inhibits the Inflammatory Response"

    Article Title: Differential Expression of microRNAs in Francisella tularensis-Infected Human Macrophages: miR-155-Dependent Downregulation of MyD88 Inhibits the Inflammatory Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109525

    Validation of the TLDA results by qRT-PCR analysis. The relative abundance of differentially expressed microRNAs was determined using individual Taqman microRNA assays, and results were normalized to the internal control RNU48. Data shown are the mean ±SEM from four independent experiments.
    Figure Legend Snippet: Validation of the TLDA results by qRT-PCR analysis. The relative abundance of differentially expressed microRNAs was determined using individual Taqman microRNA assays, and results were normalized to the internal control RNU48. Data shown are the mean ±SEM from four independent experiments.

    Techniques Used: TLDA Assay, Quantitative RT-PCR

    19) Product Images from "Diesel Exhaust Particles Activate the Matrix-Metalloproteinase-1 Gene in Human Bronchial Epithelia in a ?-Arrestin-Dependent Manner via Activation of RAS"

    Article Title: Diesel Exhaust Particles Activate the Matrix-Metalloproteinase-1 Gene in Human Bronchial Epithelia in a ?-Arrestin-Dependent Manner via Activation of RAS

    Journal: Environmental Health Perspectives

    doi: 10.1289/ehp.0800311

    DEP MMP-1 response increases in an allele-specific manner for the human–1607G(G) MMP-1 promoter polymorphism. ( A ) In BEAS-2B cells exposed to DEPs (100 μg/mL), MMP-1 mRNA formation is increased (vs. P90 nanoparticle control) as evidenced by relative abundance of the MMP-1 transcript, determined by Taqman real-time qRT-PCR. Statistically significant up-regulation of MMP-1 mRNA; experiment conducted in triplicate. ( B ) A 4.4-kb MMP-1 promoter reporter gene construct was transfected into BEAS-2B cells. Schematic illustrates the promoter with transcription factor binding sites (left). The large arrow denotes the –1607G(G) human polymorphism, which generates an ETS transcription factor binding site for GG; two smaller arrows denote additional upstream ETS binding sites. Diagram at right illustrates MMP-1 –fLUC reporter gene activity (normalized for Renilla ), in relative units (RU). Note that in response to DEPs (100 μg/mL; 24-hr incubation; triplicate assays; data based on three or more experiments), fLUC activity was strikingly increased for –1607GG (2G). ( C ) DNA sequencing of the MMP-1 promoter, encompassing –1607, from BEAS-2B cells. Like approximately 50% of all humans, BEAS-2B cells have a heterozygous genotype (–1607GG; –1607G). In ( A ), * p
    Figure Legend Snippet: DEP MMP-1 response increases in an allele-specific manner for the human–1607G(G) MMP-1 promoter polymorphism. ( A ) In BEAS-2B cells exposed to DEPs (100 μg/mL), MMP-1 mRNA formation is increased (vs. P90 nanoparticle control) as evidenced by relative abundance of the MMP-1 transcript, determined by Taqman real-time qRT-PCR. Statistically significant up-regulation of MMP-1 mRNA; experiment conducted in triplicate. ( B ) A 4.4-kb MMP-1 promoter reporter gene construct was transfected into BEAS-2B cells. Schematic illustrates the promoter with transcription factor binding sites (left). The large arrow denotes the –1607G(G) human polymorphism, which generates an ETS transcription factor binding site for GG; two smaller arrows denote additional upstream ETS binding sites. Diagram at right illustrates MMP-1 –fLUC reporter gene activity (normalized for Renilla ), in relative units (RU). Note that in response to DEPs (100 μg/mL; 24-hr incubation; triplicate assays; data based on three or more experiments), fLUC activity was strikingly increased for –1607GG (2G). ( C ) DNA sequencing of the MMP-1 promoter, encompassing –1607, from BEAS-2B cells. Like approximately 50% of all humans, BEAS-2B cells have a heterozygous genotype (–1607GG; –1607G). In ( A ), * p

    Techniques Used: Quantitative RT-PCR, Construct, Transfection, Binding Assay, Activity Assay, Incubation, DNA Sequencing

    20) Product Images from "Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer"

    Article Title: Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2015.201

    Expression of six microRNAs in the miR-17-92 cluster. Quantitative RT–PCR using Taqman miRNA assays was used to investigate the expression of the six miRNAs in exosomes purified from serum. The obtained values were normalised to hsa-miR-16a as an internal control. ( A ) Expression of 6 serum exosomal miRNAs in 6 healthy volunteers and 90 patients with CRC. ( B ) Expression of serum exosomal miR-19a in healthy individuals and patients with different stages of CRC. ( C ) Expression of serum exosomal miR-92a in healthy individuals and patients with different stages of CRC.
    Figure Legend Snippet: Expression of six microRNAs in the miR-17-92 cluster. Quantitative RT–PCR using Taqman miRNA assays was used to investigate the expression of the six miRNAs in exosomes purified from serum. The obtained values were normalised to hsa-miR-16a as an internal control. ( A ) Expression of 6 serum exosomal miRNAs in 6 healthy volunteers and 90 patients with CRC. ( B ) Expression of serum exosomal miR-19a in healthy individuals and patients with different stages of CRC. ( C ) Expression of serum exosomal miR-92a in healthy individuals and patients with different stages of CRC.

    Techniques Used: Expressing, Quantitative RT-PCR, Purification

    21) Product Images from "High glucose, unsaturated and saturated fatty acids differentially regulate expression of ATP-binding cassette transporters ABCA1 and ABCG1 in human macrophages"

    Article Title: High glucose, unsaturated and saturated fatty acids differentially regulate expression of ATP-binding cassette transporters ABCA1 and ABCG1 in human macrophages

    Journal: Experimental & Molecular Medicine

    doi: 10.3858/emm.2009.41.2.015

    ABCA1 and ABCG1 mRNA levels in human monocyte-derived macrophages as determined by Taqman real-time qRT-PCR. (A) Time kinetic experiments with normal human blood donor macrophages incubated in the presence of M-CSF for the indicated time points. (B) mRNA
    Figure Legend Snippet: ABCA1 and ABCG1 mRNA levels in human monocyte-derived macrophages as determined by Taqman real-time qRT-PCR. (A) Time kinetic experiments with normal human blood donor macrophages incubated in the presence of M-CSF for the indicated time points. (B) mRNA

    Techniques Used: Derivative Assay, Quantitative RT-PCR, Incubation

    22) Product Images from "Loss of Foxd3 Results in Decreased ?-Cell Proliferation and Glucose Intolerance During Pregnancy"

    Article Title: Loss of Foxd3 Results in Decreased ?-Cell Proliferation and Glucose Intolerance During Pregnancy

    Journal: Endocrinology

    doi: 10.1210/en.2010-1462

    Gene expression analyses by TaqMan low density arrays and quantitative real-time PCR (qRT-PCR)
    Figure Legend Snippet: Gene expression analyses by TaqMan low density arrays and quantitative real-time PCR (qRT-PCR)

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

    23) Product Images from "PCR-based allelic discrimination for glucose-6-phosphate dehydrogenase (G6PD) deficiency in Ugandan umbilical cord blood"

    Article Title: PCR-based allelic discrimination for glucose-6-phosphate dehydrogenase (G6PD) deficiency in Ugandan umbilical cord blood

    Journal: Pediatric hematology and oncology

    doi: 10.3109/08880018.2013.860649

    Examples of results from Taqman based allele specific PCR
    Figure Legend Snippet: Examples of results from Taqman based allele specific PCR

    Techniques Used: Polymerase Chain Reaction

    24) Product Images from "Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma"

    Article Title: Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2012.525

    Comparison of the serum levels of miR-720 ( A ), miR-1308 ( B ) and miR-1246 ( C ) in Normal (N), Normal hospitalised (NH), MGUS (MG) and myeloma (M) groups. Graphs show median level with interquartile range. The Kruskal–Wallis test with Dunn's post test was used to determine the significance of differences between groups. The serum levels were determined using TaqMan miRNA qRT–PCR following RNA extraction. Two technical replicates were performed on two cDNA replicates (four technical replicates total per sample/miRNA combination.
    Figure Legend Snippet: Comparison of the serum levels of miR-720 ( A ), miR-1308 ( B ) and miR-1246 ( C ) in Normal (N), Normal hospitalised (NH), MGUS (MG) and myeloma (M) groups. Graphs show median level with interquartile range. The Kruskal–Wallis test with Dunn's post test was used to determine the significance of differences between groups. The serum levels were determined using TaqMan miRNA qRT–PCR following RNA extraction. Two technical replicates were performed on two cDNA replicates (four technical replicates total per sample/miRNA combination.

    Techniques Used: Quantitative RT-PCR, RNA Extraction

    25) Product Images from "Multi-platform assessment of transcriptional profiling technologies utilizing a precise probe mapping methodology"

    Article Title: Multi-platform assessment of transcriptional profiling technologies utilizing a precise probe mapping methodology

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1913-6

    TaqMan qRT-PCR validation. ( a - f ) fold-change data derived from titrated samples. 6 genes were selected because the TaqMan probes targeted the same transcript pattern as did the microarray probe(set)s; ( g ) mean fold-change of the 6 genes across platforms; extreme values in ( a - d ) and ( g ) were indicated with broken y-axis and actual data; ( h ) mean fold-change of the 4 titrations across platforms; (I) Concordance of fold-change between TaqMan qPCR (X) and microarrays/RNA-seq protocols (Y), 4 different calls were made: compress, opposite, overestimate, and concordant. When two compared fold-changes are in the same direction but the ratio of X/Y greater than or equal to 2, a value of “compressed” is assigned. Similarly, if the fold-change ratio of X/Y is less than or equal to 0.5 the comparison is deemed “overestimate”. Fold-change ratios between these values are deemed “concordant”. When two fold-changes are not in the same direction and either of them is greater than 2 or less than 0.5, the comparison is determined to be “opposite”. Concordance rates were calculated by number of genes with “concordant” and “overestimate” calls divided by the total genes analyzed
    Figure Legend Snippet: TaqMan qRT-PCR validation. ( a - f ) fold-change data derived from titrated samples. 6 genes were selected because the TaqMan probes targeted the same transcript pattern as did the microarray probe(set)s; ( g ) mean fold-change of the 6 genes across platforms; extreme values in ( a - d ) and ( g ) were indicated with broken y-axis and actual data; ( h ) mean fold-change of the 4 titrations across platforms; (I) Concordance of fold-change between TaqMan qPCR (X) and microarrays/RNA-seq protocols (Y), 4 different calls were made: compress, opposite, overestimate, and concordant. When two compared fold-changes are in the same direction but the ratio of X/Y greater than or equal to 2, a value of “compressed” is assigned. Similarly, if the fold-change ratio of X/Y is less than or equal to 0.5 the comparison is deemed “overestimate”. Fold-change ratios between these values are deemed “concordant”. When two fold-changes are not in the same direction and either of them is greater than 2 or less than 0.5, the comparison is determined to be “opposite”. Concordance rates were calculated by number of genes with “concordant” and “overestimate” calls divided by the total genes analyzed

    Techniques Used: Quantitative RT-PCR, Derivative Assay, Microarray, Real-time Polymerase Chain Reaction, RNA Sequencing Assay

    26) Product Images from "Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase"

    Article Title: Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2016.38

    Transcriptionally regulation and transposition mediated by Sleeping Beauty (SB) transposase carried by IDLV vectors. ( a ) qRT-PCR assay on HeLa cells transduced with IDLVTKSB (2,600 ng p24) in presence or in absence of IDLVrtTA2 S -M2 (9,600 ng p24) and dox. Transposase expression upon activation was measured by real time Taqman RT-PCR and plotted as RQ value with respect to the off state (IDLVTKSB alone, RQ value = 1). The experiment was performed in triplicate and is presented the mean ± SEM. Dashed lines connect samples showing significant different values (*** P
    Figure Legend Snippet: Transcriptionally regulation and transposition mediated by Sleeping Beauty (SB) transposase carried by IDLV vectors. ( a ) qRT-PCR assay on HeLa cells transduced with IDLVTKSB (2,600 ng p24) in presence or in absence of IDLVrtTA2 S -M2 (9,600 ng p24) and dox. Transposase expression upon activation was measured by real time Taqman RT-PCR and plotted as RQ value with respect to the off state (IDLVTKSB alone, RQ value = 1). The experiment was performed in triplicate and is presented the mean ± SEM. Dashed lines connect samples showing significant different values (*** P

    Techniques Used: Quantitative RT-PCR, Transduction, Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction

    Sleeping Beauty (SB) transposase expression and activity from Tet-On transcriptionally regulated plasmids. (a) Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) assay on the transposase expression. HeLa cells were transfected with pTetOTKSB and pTetOCMVSB alone or in combination with pPGKrtTA2 S -M2 in presence and in absence of dox. Transposase expression was measured by Taqman RT PCR and plotted as RQ value with respect to the off state (pTetOTKSB and pTetOCMVSB alone, RQ value = 1). The experiment was performed in triplicate and is presented as mean value ± standard error of the mean (SEM). Dashed lines connect samples showing significant different values (* P
    Figure Legend Snippet: Sleeping Beauty (SB) transposase expression and activity from Tet-On transcriptionally regulated plasmids. (a) Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) assay on the transposase expression. HeLa cells were transfected with pTetOTKSB and pTetOCMVSB alone or in combination with pPGKrtTA2 S -M2 in presence and in absence of dox. Transposase expression was measured by Taqman RT PCR and plotted as RQ value with respect to the off state (pTetOTKSB and pTetOCMVSB alone, RQ value = 1). The experiment was performed in triplicate and is presented as mean value ± standard error of the mean (SEM). Dashed lines connect samples showing significant different values (* P

    Techniques Used: Expressing, Activity Assay, Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Reverse Transcription Polymerase Chain Reaction

    27) Product Images from "Aberrant Expression of Interleukin-1β and Inflammasome Activation in Human Malignant Gliomas"

    Article Title: Aberrant Expression of Interleukin-1β and Inflammasome Activation in Human Malignant Gliomas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103432

    miR-132, miR-212 or the proteasome inhibitor lactacystin do not affect the expression of IL-1 in human astrocytes. Human astrocytes were transfected with specific or control anti-miR inhibitors (10 nM) for 48 h, and then stimulated with IL-1α for 24 h. (A) The expression of miR-132 was quantified by TaqMan real-time RT-PCR. Specific anti-miRs but not control anti-miR suppress miR-132 expression. (B) The culture supernatants were examined for the presence of IL-1β protein by sensitive ELISA with a lower detection limit of 3.9 pg/ml. There was no detectable IL-1β protein production in any of the human astrocyte cultures examined. (C) The effect of the proteasome inhibitor lactacystin on astrocyte IL-1β was examined. Astrocytes were treated with lactacystin at indicated concentrations with or without IL-1α, then cell lysates were subjected to ELISA after 24 h. IL-1β protein was undetectable under any conditions. Mean ± SD from triplicate cultures.
    Figure Legend Snippet: miR-132, miR-212 or the proteasome inhibitor lactacystin do not affect the expression of IL-1 in human astrocytes. Human astrocytes were transfected with specific or control anti-miR inhibitors (10 nM) for 48 h, and then stimulated with IL-1α for 24 h. (A) The expression of miR-132 was quantified by TaqMan real-time RT-PCR. Specific anti-miRs but not control anti-miR suppress miR-132 expression. (B) The culture supernatants were examined for the presence of IL-1β protein by sensitive ELISA with a lower detection limit of 3.9 pg/ml. There was no detectable IL-1β protein production in any of the human astrocyte cultures examined. (C) The effect of the proteasome inhibitor lactacystin on astrocyte IL-1β was examined. Astrocytes were treated with lactacystin at indicated concentrations with or without IL-1α, then cell lysates were subjected to ELISA after 24 h. IL-1β protein was undetectable under any conditions. Mean ± SD from triplicate cultures.

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    28) Product Images from "Quantitative analysis of interferon alpha receptor subunit 1 and suppressor of cytokine signaling 1 gene transcription in blood cells of patients with chronic hepatitis C"

    Article Title: Quantitative analysis of interferon alpha receptor subunit 1 and suppressor of cytokine signaling 1 gene transcription in blood cells of patients with chronic hepatitis C

    Journal: Virology Journal

    doi: 10.1186/1743-422X-7-243

    Validation of ifnar1 real-time RT-PCR . Ifnar1 detection assay was based on the use of TaqMan probes. Validation curves were performed with RNA extracted from HeLa cells, and the threshold cycle difference ( ΔCT ) between ifnar1 and the constitutive endogenous gene hprt was calculated and drawn with respect to RNA concentration. Slope value ( y ) indicates that hprt can be used for relative quantification of ifnar1 transcription.
    Figure Legend Snippet: Validation of ifnar1 real-time RT-PCR . Ifnar1 detection assay was based on the use of TaqMan probes. Validation curves were performed with RNA extracted from HeLa cells, and the threshold cycle difference ( ΔCT ) between ifnar1 and the constitutive endogenous gene hprt was calculated and drawn with respect to RNA concentration. Slope value ( y ) indicates that hprt can be used for relative quantification of ifnar1 transcription.

    Techniques Used: Quantitative RT-PCR, Detection Assay, Concentration Assay

    29) Product Images from "Natural BH3-mimetic (-)-gossypol chemosensitizes human prostate cancer via Bcl-xL inhibition accompanied by increase of Puma and Noxa"

    Article Title: Natural BH3-mimetic (-)-gossypol chemosensitizes human prostate cancer via Bcl-xL inhibition accompanied by increase of Puma and Noxa

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-08-0333

    (-)-Gossypol increases Noxa, Puma, Mcl-1 and decreases Bim at protein levels. A. (-)-gossypol dose-dependently induced Mcl-1, Noxa, Puma increase and Bim decrease. B . (-)-gossypol-induced Mcl-1 increase is caspase independent. PC-3 cells were pretreated with zVAD-fmk for 4 hours, then incubated with or without (-)-gossypol for another 24 hours before collecting for Western blot analysis. C. Changes of mRNA levels of Mcl-1, Noxa and Puma. PC-3 cells were treated with (-)-gossypol at the indicated time-points and doses. qRT-PCR reactions with TaqMan Universal PCR Master Mix (Applied Biosystems) were performed on the Mastercycler realplex 2 system (Eppendorf, Westbury, NY). Target gene mRNA levels were normalized to Actin mRNA according to the following formula: [2ˆ –(C T target – C T Actin )] × 100%, where C T is the threshold cycle. Fold increase was calculated by dividing the normalized target gene expression of the treated sample with that of the untreated control.
    Figure Legend Snippet: (-)-Gossypol increases Noxa, Puma, Mcl-1 and decreases Bim at protein levels. A. (-)-gossypol dose-dependently induced Mcl-1, Noxa, Puma increase and Bim decrease. B . (-)-gossypol-induced Mcl-1 increase is caspase independent. PC-3 cells were pretreated with zVAD-fmk for 4 hours, then incubated with or without (-)-gossypol for another 24 hours before collecting for Western blot analysis. C. Changes of mRNA levels of Mcl-1, Noxa and Puma. PC-3 cells were treated with (-)-gossypol at the indicated time-points and doses. qRT-PCR reactions with TaqMan Universal PCR Master Mix (Applied Biosystems) were performed on the Mastercycler realplex 2 system (Eppendorf, Westbury, NY). Target gene mRNA levels were normalized to Actin mRNA according to the following formula: [2ˆ –(C T target – C T Actin )] × 100%, where C T is the threshold cycle. Fold increase was calculated by dividing the normalized target gene expression of the treated sample with that of the untreated control.

    Techniques Used: Incubation, Western Blot, Quantitative RT-PCR, Polymerase Chain Reaction, Expressing

    30) Product Images from "A Critical Point of Male Gonad Development: Neuroendocrine Correlates of Accelerated Testicular Growth in Rats during Early Life"

    Article Title: A Critical Point of Male Gonad Development: Neuroendocrine Correlates of Accelerated Testicular Growth in Rats during Early Life

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093007

    GnRH and its receptor mRNA levels in the hypothalamus of neonatal male rats. Probes of cDNA were synthesized from total mRNA extracted from the anterior hypothalamic preoptic area of 3–14 day-old male rats. Probes were examined for GnRH and its receptor mRNA expression by the TaqMan assay-based real-time PCR. The comparative ddCT method was used to calculate mRNA expression relative to the beta-actin as an endogenous control. Data represent mean ± SEM of 6–8 individual animals. a p
    Figure Legend Snippet: GnRH and its receptor mRNA levels in the hypothalamus of neonatal male rats. Probes of cDNA were synthesized from total mRNA extracted from the anterior hypothalamic preoptic area of 3–14 day-old male rats. Probes were examined for GnRH and its receptor mRNA expression by the TaqMan assay-based real-time PCR. The comparative ddCT method was used to calculate mRNA expression relative to the beta-actin as an endogenous control. Data represent mean ± SEM of 6–8 individual animals. a p

    Techniques Used: Synthesized, Expressing, TaqMan Assay, Real-time Polymerase Chain Reaction

    Testes weight in 7-day old rats after siRNA knockdown of GnRH expression. siRNA targeting GnRH mRNA from 369 to 389 (200 μM in 5 μl of OptiMEM) was injected into the anterior hypothalamic preoptic area on the day 5 of life. Animals of control groups received the same volume of vehicle or the same amount of control short double stranded RNA (dsRNA), which has no homology with any rat mRNA. Rats were weighted and sacrificed at the day 7 of life. Testes were dissected and weighted. Probes of cDNA were synthesized from total mRNA extracted from the anterior hypothalamic preoptic area. Probes were examined for GnRH mRNA expression by the TaqMan assay-based real-time PCR. The comparative ddCT method was used to calculate mRNA expression relative to the beta-actin as an endogenous control. Data are expressed as percentages of the values for the vehicle-treated control group that was taken as 100%, and presented as mean ± SEM of 7 individual animals. *p
    Figure Legend Snippet: Testes weight in 7-day old rats after siRNA knockdown of GnRH expression. siRNA targeting GnRH mRNA from 369 to 389 (200 μM in 5 μl of OptiMEM) was injected into the anterior hypothalamic preoptic area on the day 5 of life. Animals of control groups received the same volume of vehicle or the same amount of control short double stranded RNA (dsRNA), which has no homology with any rat mRNA. Rats were weighted and sacrificed at the day 7 of life. Testes were dissected and weighted. Probes of cDNA were synthesized from total mRNA extracted from the anterior hypothalamic preoptic area. Probes were examined for GnRH mRNA expression by the TaqMan assay-based real-time PCR. The comparative ddCT method was used to calculate mRNA expression relative to the beta-actin as an endogenous control. Data are expressed as percentages of the values for the vehicle-treated control group that was taken as 100%, and presented as mean ± SEM of 7 individual animals. *p

    Techniques Used: Expressing, Injection, Synthesized, TaqMan Assay, Real-time Polymerase Chain Reaction

    Kisspeptin and its receptor mRNA levels in the hypothalamus of neonatal male rats. Probes of cDNA were synthesized from total mRNA extracted from the anterior hypothalamic preoptic area of 3–14 day-old male rats. Probes were examined for kisspeptin and its receptor mRNA expression by the TaqMan assay-based real-time PCR. The comparative ddCT method was used to calculate mRNA expression relative to the beta-actin as an endogenous control. Data represent mean ± SEM of 3–6 individual animals. a p
    Figure Legend Snippet: Kisspeptin and its receptor mRNA levels in the hypothalamus of neonatal male rats. Probes of cDNA were synthesized from total mRNA extracted from the anterior hypothalamic preoptic area of 3–14 day-old male rats. Probes were examined for kisspeptin and its receptor mRNA expression by the TaqMan assay-based real-time PCR. The comparative ddCT method was used to calculate mRNA expression relative to the beta-actin as an endogenous control. Data represent mean ± SEM of 3–6 individual animals. a p

    Techniques Used: Synthesized, Expressing, TaqMan Assay, Real-time Polymerase Chain Reaction

    Correlation between testes weights and GnRH mRNA levels in the hypothalamus. Rats were weighted and sacrificed at the days 9, 11 and 14 of life. Testes were dissected and weighted. Probes of cDNA were synthesized from total mRNA extracted from the anterior hypothalamic preoptic area. Probes were examined for GnRH mRNA expression by the TaqMan assay-based real-time PCR. The comparative ddCT method was used to calculate mRNA expression relative to the beta-actin as an endogenous control. Each point represents an individual animal. Significant Pearson's linear correlation (r = 0.689 p
    Figure Legend Snippet: Correlation between testes weights and GnRH mRNA levels in the hypothalamus. Rats were weighted and sacrificed at the days 9, 11 and 14 of life. Testes were dissected and weighted. Probes of cDNA were synthesized from total mRNA extracted from the anterior hypothalamic preoptic area. Probes were examined for GnRH mRNA expression by the TaqMan assay-based real-time PCR. The comparative ddCT method was used to calculate mRNA expression relative to the beta-actin as an endogenous control. Each point represents an individual animal. Significant Pearson's linear correlation (r = 0.689 p

    Techniques Used: Synthesized, Expressing, TaqMan Assay, Real-time Polymerase Chain Reaction

    31) Product Images from "Adaptive Evolution of a Tagged Chimeric Gammaretrovirus: Identification of Novel cis-Acting Elements That Modulate Splicing"

    Article Title: Adaptive Evolution of a Tagged Chimeric Gammaretrovirus: Identification of Novel cis-Acting Elements That Modulate Splicing

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2007.04.026

    Quantitation of the levels of spliced and unspliced viral RNA produced by MLV, GALV and chimeric viruses. RNA isolated from cells transfected with the provirus-containing plasmids was reverse transcribed and used in TaqMan PCR. (a) Schematic diagram of
    Figure Legend Snippet: Quantitation of the levels of spliced and unspliced viral RNA produced by MLV, GALV and chimeric viruses. RNA isolated from cells transfected with the provirus-containing plasmids was reverse transcribed and used in TaqMan PCR. (a) Schematic diagram of

    Techniques Used: Quantitation Assay, Produced, Isolation, Transfection, Polymerase Chain Reaction

    32) Product Images from "Treponema denticola TroR is a manganese- and iron-dependent transcriptional repressor"

    Article Title: Treponema denticola TroR is a manganese- and iron-dependent transcriptional repressor

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2008.06418.x

    T. denticola tro operon is negatively regulated by Mn 2+ and Fe 2+ . Expression of troA was analysed by qRT-PCR. RNA extracted from T. denticola grown in NOS-EC media supplemented with 5 μM Fe 2+ , Mn 2+ or Zn 2+ was quantified at (A) 24 h or (B) 48 h post inoculation using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaA . Fold changes are relative to spirochaetes grown in NOS-EC media lacking metal supplmentation (control). Results represent the means and standard deviations of three independent experiments performed in quadruplicate.
    Figure Legend Snippet: T. denticola tro operon is negatively regulated by Mn 2+ and Fe 2+ . Expression of troA was analysed by qRT-PCR. RNA extracted from T. denticola grown in NOS-EC media supplemented with 5 μM Fe 2+ , Mn 2+ or Zn 2+ was quantified at (A) 24 h or (B) 48 h post inoculation using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaA . Fold changes are relative to spirochaetes grown in NOS-EC media lacking metal supplmentation (control). Results represent the means and standard deviations of three independent experiments performed in quadruplicate.

    Techniques Used: Expressing, Quantitative RT-PCR

    33) Product Images from "Human CYP2C8 Is Post-Transcriptionally Regulated by MicroRNAs 103 and 107 in Human Liver"

    Article Title: Human CYP2C8 Is Post-Transcriptionally Regulated by MicroRNAs 103 and 107 in Human Liver

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.112.078386

    Human CYP2C8 regulation by miR-103/miR-107. A, relative endogenous expression levels of miR-103 and miR-107 in primary hepatocytes (PH) and HepG2 cells were determined through real-time quantitative PCR analysis using a TaqMan miRNA assay kit, and the levels of mature miR-103 and miR-107 were normalized with respect to RNU44 levels with the 2 −ΔΔCt method. B and C, luciferase assays were performed to investigate whether miR-103 and miR-107 functionally regulated CYP2C8MRE. The reporter constructs were transiently transfected into primary human hepatocytes with 10 pmol of the precursors for miR-103 or miR-107 or control precursors (B) or with 10 pmol of AsOs for miR-103 or miR-107 or AsO controls (C). pMIR-driven firefly luciferase activities were normalized with respect to R. reniformis luciferase activities and were plotted relative to values obtained with the pMIR control vector. Each column represents the mean ± S.E. of three independent experiments. *, significantly different from precursor or AsO control, P
    Figure Legend Snippet: Human CYP2C8 regulation by miR-103/miR-107. A, relative endogenous expression levels of miR-103 and miR-107 in primary hepatocytes (PH) and HepG2 cells were determined through real-time quantitative PCR analysis using a TaqMan miRNA assay kit, and the levels of mature miR-103 and miR-107 were normalized with respect to RNU44 levels with the 2 −ΔΔCt method. B and C, luciferase assays were performed to investigate whether miR-103 and miR-107 functionally regulated CYP2C8MRE. The reporter constructs were transiently transfected into primary human hepatocytes with 10 pmol of the precursors for miR-103 or miR-107 or control precursors (B) or with 10 pmol of AsOs for miR-103 or miR-107 or AsO controls (C). pMIR-driven firefly luciferase activities were normalized with respect to R. reniformis luciferase activities and were plotted relative to values obtained with the pMIR control vector. Each column represents the mean ± S.E. of three independent experiments. *, significantly different from precursor or AsO control, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, TaqMan microRNA Assay, Luciferase, Construct, Transfection, Allele-specific Oligonucleotide, Plasmid Preparation

    34) Product Images from "miR-203 is involved in the laryngeal carcinoma pathogenesis via targeting VEGFA and Cox-2"

    Article Title: miR-203 is involved in the laryngeal carcinoma pathogenesis via targeting VEGFA and Cox-2

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S96053

    The expression of miR-203 was downregulated in LSCC tissues. Notes: Total RNA was extracted from tissues samples by using TRIzol reagent according to the manufacturer’s instructions. Random six RNA samples were mixed as one sample for the detection of target molecules. The expression of miRNAs was detected by TaqMan miRNA RT-Real-Time PCR. The U6 snRNA was used for normalization. Each sample in each group was measured in triplicate, and the experiment was repeated at least three times for the detection of miRNAs. Student’s t -test was used to analyze the expression of candidate miRNAs in tumor and nontumor groups, and P
    Figure Legend Snippet: The expression of miR-203 was downregulated in LSCC tissues. Notes: Total RNA was extracted from tissues samples by using TRIzol reagent according to the manufacturer’s instructions. Random six RNA samples were mixed as one sample for the detection of target molecules. The expression of miRNAs was detected by TaqMan miRNA RT-Real-Time PCR. The U6 snRNA was used for normalization. Each sample in each group was measured in triplicate, and the experiment was repeated at least three times for the detection of miRNAs. Student’s t -test was used to analyze the expression of candidate miRNAs in tumor and nontumor groups, and P

    Techniques Used: Expressing, miRNA RT, Real-time Polymerase Chain Reaction

    35) Product Images from "Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment"

    Article Title: Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2010.5

    Anti-miRNA inhibitor molecule transfections. ( a ) TaqMan real-time PCR evaluation of mature hsa-miR-299-5p microRNA expression after 24, 72 and 96 h of cell culture in the presence of TPO (100 ng/ml). Data were obtained using as calibrator the RNA of the same sample extracted immediately after CD34 HPC purification and RNU6b as endogenous control. Cells were electroporated with hsa-miR-299-5p inhibitor after 3 days of culture. ( b ) TaqMan real-time PCR mature hsa-miR-299-5p expression after single transient transfection of its inhibitor molecule, through single nucleoporation with the Amaxa Nucleofector device. Expression withdrawal was monitored after one and ten days from nucleofection. Data were obtained using as calibrator the nucleofection solution-treated sample (nuc) and RNU6b as endogenous control. ( c ) Colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA inhibitor (anti-299-5p), compared with Anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001. BFU-E: erythrocyte burst-forming unit; CFU-E: erythrocyte colony-forming unit; GM-CFU: granulo-monocyte colony-forming unit; G-CFU: granulocyte colony-forming unit; M-CFU: monocyte colony-forming unit; GEMM-CFU: granulocyte-erythrocyte-monocyte-macrophage colony-forming unit. ( d ) Megakaryocytic colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA Inhibitor (anti-299-5p), compared with anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001
    Figure Legend Snippet: Anti-miRNA inhibitor molecule transfections. ( a ) TaqMan real-time PCR evaluation of mature hsa-miR-299-5p microRNA expression after 24, 72 and 96 h of cell culture in the presence of TPO (100 ng/ml). Data were obtained using as calibrator the RNA of the same sample extracted immediately after CD34 HPC purification and RNU6b as endogenous control. Cells were electroporated with hsa-miR-299-5p inhibitor after 3 days of culture. ( b ) TaqMan real-time PCR mature hsa-miR-299-5p expression after single transient transfection of its inhibitor molecule, through single nucleoporation with the Amaxa Nucleofector device. Expression withdrawal was monitored after one and ten days from nucleofection. Data were obtained using as calibrator the nucleofection solution-treated sample (nuc) and RNU6b as endogenous control. ( c ) Colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA inhibitor (anti-299-5p), compared with Anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001. BFU-E: erythrocyte burst-forming unit; CFU-E: erythrocyte colony-forming unit; GM-CFU: granulo-monocyte colony-forming unit; G-CFU: granulocyte colony-forming unit; M-CFU: monocyte colony-forming unit; GEMM-CFU: granulocyte-erythrocyte-monocyte-macrophage colony-forming unit. ( d ) Megakaryocytic colony-forming capacity of HPCs transfected with hsa-miR-299-5p Anti-miR miRNA Inhibitor (anti-299-5p), compared with anti-miR miRNA inhibitor-negative control #1 (anti-NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from three independent experiments. Asterisk indicates a t -test P -value⩽0.001

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Purification, Negative Control

    Pre-miR miRNA Precursor Molecule transfections. ( a ) TaqMan real-time PCR of mature hsa-miR-299-5p expression after single transient transfection of its precursor molecule through the Amaxa Nucleofector device. Hsa-miR-299-5p expression was monitored at different time points during in vitro culture using the nucleofection solution-treated sample (nuc) as calibrator and RNU6b as endogenous control. ( b ) Cytofluorimetric evaluation of the differentiation surface markers' expression: CD235a for erythroid differentiation, CD41a as indicator of megakaryocytic commitment, CD14 distinguishing monocytic differentiation and CD66b reflecting granulocytic cell fate. The surface markers' expression was checked after seven days of culture and each bar represents the fold change of the percentage of positivity normalized onto the pre-miR miRNA precursor molecule-negative control # 1 (NC1)-treated sample. Asterisk indicates a t -test P -value ⩽0.001. ( c ) Colony-forming capacity of HPCs transfected with pre-miR miRNA precursor molecule (299-5p), when compared with Pre-miR miRNA Precursor molecule-negative control # 1 (NC1)-transfected cells and with cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from four independent experiments. BFU-E: erythrocyte burst-forming unit; CFU-E: erythrocyte colony-forming unit; GM-CFU: granulo-monocyte colony-forming unit; G-CFU: granulocyte colony-forming unit; M-CFU: monocyte colony-forming unit; GEMM-CFU: granulocyte-erythrocyte-monocyte-macrophage colony-forming unit. Asterisk indicates a t -test P -value ⩽0.001. ( d ) Megakaryocytic colony-forming capacity of HPCs transfected with pre-miR miRNA precursor molecule (299-5p), when compared with pre-miR miRNA precursor molecule-negative control # 1 (NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from four independent experiments. Asterisk indicates a t -test P -value ⩽0.001
    Figure Legend Snippet: Pre-miR miRNA Precursor Molecule transfections. ( a ) TaqMan real-time PCR of mature hsa-miR-299-5p expression after single transient transfection of its precursor molecule through the Amaxa Nucleofector device. Hsa-miR-299-5p expression was monitored at different time points during in vitro culture using the nucleofection solution-treated sample (nuc) as calibrator and RNU6b as endogenous control. ( b ) Cytofluorimetric evaluation of the differentiation surface markers' expression: CD235a for erythroid differentiation, CD41a as indicator of megakaryocytic commitment, CD14 distinguishing monocytic differentiation and CD66b reflecting granulocytic cell fate. The surface markers' expression was checked after seven days of culture and each bar represents the fold change of the percentage of positivity normalized onto the pre-miR miRNA precursor molecule-negative control # 1 (NC1)-treated sample. Asterisk indicates a t -test P -value ⩽0.001. ( c ) Colony-forming capacity of HPCs transfected with pre-miR miRNA precursor molecule (299-5p), when compared with Pre-miR miRNA Precursor molecule-negative control # 1 (NC1)-transfected cells and with cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from four independent experiments. BFU-E: erythrocyte burst-forming unit; CFU-E: erythrocyte colony-forming unit; GM-CFU: granulo-monocyte colony-forming unit; G-CFU: granulocyte colony-forming unit; M-CFU: monocyte colony-forming unit; GEMM-CFU: granulocyte-erythrocyte-monocyte-macrophage colony-forming unit. Asterisk indicates a t -test P -value ⩽0.001. ( d ) Megakaryocytic colony-forming capacity of HPCs transfected with pre-miR miRNA precursor molecule (299-5p), when compared with pre-miR miRNA precursor molecule-negative control # 1 (NC1)-transfected cells and cells electroporated with the nucleofection solution only (nuc). Mean±S.D. from four independent experiments. Asterisk indicates a t -test P -value ⩽0.001

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Expressing, In Vitro, Negative Control

    MicroRNAs oppositely regulated during myeloid differentiation. Twenty-three microRNAs (indicated with the prefix miR-) show either up- or downregulation greater than four in at least one precursor population when compared with the CD34 HPCs; 17 showed a significant increased expression with an RQ higher than four, 15 a significant decreased expression with an RQ lower than −4 and concomitantly a counter-regulation in at least one other precursor cell context. TaqMan real-time PCR reactions were carried out using the TaqMan MicroRNA Assays Human Panel Early Access Kit. Delta-delta-CTs and RQs were calculated for each detector using the CD34 HPC sample as the calibrator and hsa-miR-let-7a as the endogenous control
    Figure Legend Snippet: MicroRNAs oppositely regulated during myeloid differentiation. Twenty-three microRNAs (indicated with the prefix miR-) show either up- or downregulation greater than four in at least one precursor population when compared with the CD34 HPCs; 17 showed a significant increased expression with an RQ higher than four, 15 a significant decreased expression with an RQ lower than −4 and concomitantly a counter-regulation in at least one other precursor cell context. TaqMan real-time PCR reactions were carried out using the TaqMan MicroRNA Assays Human Panel Early Access Kit. Delta-delta-CTs and RQs were calculated for each detector using the CD34 HPC sample as the calibrator and hsa-miR-let-7a as the endogenous control

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    36) Product Images from "Reducing prohibitin increases histone acetylation, and promotes androgen independence in prostate tumours by increasing androgen receptor activation by adrenal androgens"

    Article Title: Reducing prohibitin increases histone acetylation, and promotes androgen independence in prostate tumours by increasing androgen receptor activation by adrenal androgens

    Journal: Oncogene

    doi: 10.1038/onc.2011.591

    Analysis of gene expression in LNCaP cell lines with altered PHB levels. (a) Taqman RT-PCR analysis of PSA transcript levels collected at time intervals (0 – 8hr) from starved LNCaP/Luc/PHB-RNAi cells treated with 10nM DHT (left hand side) or 10nM androstenedione (right hand side) with or without doxycycline. (b) Taqman RT-PCR analysis of PSA transcript levels from starved LNCaP/Luc/PHB-cDNA treated with increasing concentrations of DHT or androstenedione. (c) Taqman RT-PCR analysis of PSA transcript levels from starved LNCaP/Luc/PHB-RNAi cells treated with increasing concentrations of DHT or androstenedione (± doxycycline). (d) Luciferase activity from LNCaP/Luc/PHB-RNAi cells treated with DHT (0-100nM) or Adione (0-100nM) with or without doxycycline. Data are mean ± SD of 3 independent experiments performed in triplicate on a representative LNCaP/Luc/PHB-RNAi clone.
    Figure Legend Snippet: Analysis of gene expression in LNCaP cell lines with altered PHB levels. (a) Taqman RT-PCR analysis of PSA transcript levels collected at time intervals (0 – 8hr) from starved LNCaP/Luc/PHB-RNAi cells treated with 10nM DHT (left hand side) or 10nM androstenedione (right hand side) with or without doxycycline. (b) Taqman RT-PCR analysis of PSA transcript levels from starved LNCaP/Luc/PHB-cDNA treated with increasing concentrations of DHT or androstenedione. (c) Taqman RT-PCR analysis of PSA transcript levels from starved LNCaP/Luc/PHB-RNAi cells treated with increasing concentrations of DHT or androstenedione (± doxycycline). (d) Luciferase activity from LNCaP/Luc/PHB-RNAi cells treated with DHT (0-100nM) or Adione (0-100nM) with or without doxycycline. Data are mean ± SD of 3 independent experiments performed in triplicate on a representative LNCaP/Luc/PHB-RNAi clone.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay

    ChIP analysis of AR and PHB on the PSA regulatory region. (a) Diagramatic representation of the PSA promoter indicating locations of ARE I, II, III and the intervening regions (negative and up/down-stream). Labelling boxes indicate amplification regions of primer pairs used for PCR. DNA immunoprecipitated with either IgG control, AR or PHB antibody was amplified by PCR and the results for each region are shown underneath, compared to their respective input DNA control. Densitometry data for each band for PHB are given underneath. (b) ChIP analysis of AR and PHB binding to the PSA promoter of LNCaP cells either grown in full serum, or hormone-starved with ±10nM DHT and for 2hr. Data represents Taqman quantification of immunoprecipitated DNA, from three replicate experiments, normalised to their input DNA controls. (c) ChIP analysis of AR binding to the PSA promoter in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). (d) ChIP analysis of PHB binding to the PSA promoter in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). Data are from a representative LNCaP/Luc/PHB-RNAi clone. ** = P
    Figure Legend Snippet: ChIP analysis of AR and PHB on the PSA regulatory region. (a) Diagramatic representation of the PSA promoter indicating locations of ARE I, II, III and the intervening regions (negative and up/down-stream). Labelling boxes indicate amplification regions of primer pairs used for PCR. DNA immunoprecipitated with either IgG control, AR or PHB antibody was amplified by PCR and the results for each region are shown underneath, compared to their respective input DNA control. Densitometry data for each band for PHB are given underneath. (b) ChIP analysis of AR and PHB binding to the PSA promoter of LNCaP cells either grown in full serum, or hormone-starved with ±10nM DHT and for 2hr. Data represents Taqman quantification of immunoprecipitated DNA, from three replicate experiments, normalised to their input DNA controls. (c) ChIP analysis of AR binding to the PSA promoter in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). (d) ChIP analysis of PHB binding to the PSA promoter in LNCaP/Luc/PHB-RNAi cells after treatment with DHT for 0-2hrs (± doxycycline). Data are from a representative LNCaP/Luc/PHB-RNAi clone. ** = P

    Techniques Used: Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Immunoprecipitation, Binding Assay

    37) Product Images from "Growth factor restriction impedes progression of wound healing following cataract surgery: identification of VEGF as a putative therapeutic target"

    Article Title: Growth factor restriction impedes progression of wound healing following cataract surgery: identification of VEGF as a putative therapeutic target

    Journal: Scientific Reports

    doi: 10.1038/srep24453

    The influence of VEGF receptor inhibition using Axitinib (10 μM) on EMT/fibrotic marker genes ( a ) ACTA2 ( b ) FN1 and ( c ) MMP2 in FHL 124 cells. Cells were maintained in experimental conditions for 24 hours and gene expression was detected using TaqMan real-time PCR. Expression of the gene of interest was normalised to 18S expression. The data represent Mean ± SEM ( n = 3). *indicates a significant difference between treatment and control group ((P ≤ 0.05, Students t test).
    Figure Legend Snippet: The influence of VEGF receptor inhibition using Axitinib (10 μM) on EMT/fibrotic marker genes ( a ) ACTA2 ( b ) FN1 and ( c ) MMP2 in FHL 124 cells. Cells were maintained in experimental conditions for 24 hours and gene expression was detected using TaqMan real-time PCR. Expression of the gene of interest was normalised to 18S expression. The data represent Mean ± SEM ( n = 3). *indicates a significant difference between treatment and control group ((P ≤ 0.05, Students t test).

    Techniques Used: Inhibition, Marker, Expressing, Real-time Polymerase Chain Reaction

    38) Product Images from "Downregulation of ATG14 by EGR1-MIR152 sensitizes ovarian cancer cells to cisplatin-induced apoptosis by inhibiting cyto-protective autophagy"

    Article Title: Downregulation of ATG14 by EGR1-MIR152 sensitizes ovarian cancer cells to cisplatin-induced apoptosis by inhibiting cyto-protective autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2015.1009781

    The MIR152 mimic sensitizes ovarian cancer cells to cisplatin-mediated cell death. ( A ) MIR152 expression levels in A2780/CP70, A2780, SKOV3 and SKOV3/DDP cells were determined by Taqman RT-PCR. ( B ) Cells were transfected with the MIR152 mimic or miR-control
    Figure Legend Snippet: The MIR152 mimic sensitizes ovarian cancer cells to cisplatin-mediated cell death. ( A ) MIR152 expression levels in A2780/CP70, A2780, SKOV3 and SKOV3/DDP cells were determined by Taqman RT-PCR. ( B ) Cells were transfected with the MIR152 mimic or miR-control

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

    39) Product Images from "Makorin Ring Zinc Finger Protein 1 (MKRN1), a Novel Poly(A)-binding Protein-interacting Protein, Stimulates Translation in Nerve Cells"

    Article Title: Makorin Ring Zinc Finger Protein 1 (MKRN1), a Novel Poly(A)-binding Protein-interacting Protein, Stimulates Translation in Nerve Cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.315291

    MKRN1-short stimulates translation in nerve cells. The eukaryotic expression vector pinFiRein-boxB16B was co-transfected with vectors encoding fusion proteins consisting of an N-terminal N22 peptide and either hMKRN1-short, hMKRN1-Δ487–535, hDDX6, or rShank3–1-299 into dispersed cortical neurons at 7 DIV. The empty vector N22-FLAG3 served as a control (for details see “Experimental Procedures”). A , dual luciferase assays were performed at 9 DIV. The relative levels of PhoLuc/RenLuc proteins are shown (in arbitrary units). B , RNAs from transfected neurons were prepared on 9 DIV, transcribed into cDNAs, and subjected to real-time PCR analyses using Pho/Luc- and RenLuc-specific TaqMan assays. The relative levels of PhoLuc/RenLuc mRNAs are shown (in arbitrary units). Bars represent S.E. Statistical analyses were done using Student's t test (**, p
    Figure Legend Snippet: MKRN1-short stimulates translation in nerve cells. The eukaryotic expression vector pinFiRein-boxB16B was co-transfected with vectors encoding fusion proteins consisting of an N-terminal N22 peptide and either hMKRN1-short, hMKRN1-Δ487–535, hDDX6, or rShank3–1-299 into dispersed cortical neurons at 7 DIV. The empty vector N22-FLAG3 served as a control (for details see “Experimental Procedures”). A , dual luciferase assays were performed at 9 DIV. The relative levels of PhoLuc/RenLuc proteins are shown (in arbitrary units). B , RNAs from transfected neurons were prepared on 9 DIV, transcribed into cDNAs, and subjected to real-time PCR analyses using Pho/Luc- and RenLuc-specific TaqMan assays. The relative levels of PhoLuc/RenLuc mRNAs are shown (in arbitrary units). Bars represent S.E. Statistical analyses were done using Student's t test (**, p

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Luciferase, Real-time Polymerase Chain Reaction

    40) Product Images from "Reconstruction of liver organoid using a bioreactor"

    Article Title: Reconstruction of liver organoid using a bioreactor

    Journal:

    doi: 10.3748/wjg.v12.i12.1881

    Comparison of expressions of CPS1, OTC, ASS, ASL, and ARG mRNA in FLC-5 incubated under different conditions as assessed by TaqMan 1-step RT-PCR. The mRNA expression of each enzyme in different conditions is relative to that in monolayer cultures. Mean
    Figure Legend Snippet: Comparison of expressions of CPS1, OTC, ASS, ASL, and ARG mRNA in FLC-5 incubated under different conditions as assessed by TaqMan 1-step RT-PCR. The mRNA expression of each enzyme in different conditions is relative to that in monolayer cultures. Mean

    Techniques Used: Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Virlaza™ Inhibits Sars-COV-2-induced Inflammatory Response of Bronchial Epithelial Cells and Pulmonary Fibroblast
    Article Snippet: .. Quantitative PCR used Taqman Universal PCR Mastermix (Applied Biosystems, USA) and preformulated primers and probe mixes (Assay on Demand, Applied Biosystems, USA). ..

    Article Title: Diverse effects of pan-ROCK and ROCK2 inhibitors on 2 D and 3D cultured human trabecular meshwork (HTM) cells treated with TGFβ2
    Article Snippet: .. Total RNA extraction, reverse transcription and following real-time PCR with the Universal Taqman Master mix using a StepOnePlus instrument (Applied Biosystems/Thermo Fisher Scientific) were performed as describe previously . ..

    Polymerase Chain Reaction:

    Article Title: Virlaza™ Inhibits Sars-COV-2-induced Inflammatory Response of Bronchial Epithelial Cells and Pulmonary Fibroblast
    Article Snippet: .. Quantitative PCR used Taqman Universal PCR Mastermix (Applied Biosystems, USA) and preformulated primers and probe mixes (Assay on Demand, Applied Biosystems, USA). ..

    Article Title: Genetic and neurophysiological biomarkers of neuroplasticity inform post-stroke language recovery
    Article Snippet: .. Primers and probes were mixed with TaqMan® Universal PCR Master Mix (Thermo Fisher Scientific). ..

    Article Title: Streptococcus pneumoniae colonisation associates with impaired adaptive immune responses against SARS-CoV-2
    Article Snippet: .. Briefly, 20μL PCR mix consisted of 12.5μL 1 × TaqMan Universal PCR Master Mix (Life Technologies Ltd, Paisley, UK), 0.225μL or 0.2μL 100μ M each lytA or piaB primer respectively, 0.125μLor 0.175μL 100μM lytA or piaB probe respectively, molecular graded water (Fisher Scientific, Loughborough, UK) and 2.5μL of the extracted DNA. ..

    Article Title: Hyperosmolarity-Induced Cornification of Human Corneal Epithelial Cells Is Regulated by JNK MAPK
    Article Snippet: .. Alexa Fluor 488–conjugated goat IgG secondary antibodies and fluorescein cadaverine (F-CDV) from Invitrogen-Molecular Probes (Eugene, OR); Hoechst 33342, propidium iodide (PI), and DNA size markers from Sigma-Aldrich; RNA extraction kits (RNeasy Mini Kit, RNAi kit, and siRNA-fluorescein, siRNA-F) from Qiagen (Valencia, CA); a PCR kit (GeneAmp RNA-PCR kit and Taqman Universal PCR master mix; Applied Biosystems, Foster City, CA); a cDNA synthesis kit (Ready-To-Go-Primer First-Strand Beads) from GE Health Care, Inc. (Piscataway, NJ); nuclear and cytoplasmic extraction reagents (NE-PER) and a BCA protein assay kit from Pierce (Rockford, IL); gels for protein electrophoresis (4%–15% Tris-HCl; Ready Gel) from Bio-Rad (Hercules, CA); polyvinylidene difluoride (PVDF; Immobilon-P) membrane from Millipore (Billerica, MA); a chemiluminescent reagent (Luminol) from Santa Cruz Biotechnology (Santa Cruz, CA); an in situ TUNEL kit from Roche (Basel, Switzerland); MTT cell proliferation assay kit from R & D Systems, Inc. (Minneapolis, MN); a JNK cell signaling assay (Beadlyte) and an anti-phosphatidylserine–conjugated Alexa 488 antibody from Upstate Biotechnology (Charlottesville, VA); and an Annexin-V FITC cell viability kit from ApoTarget (Camarillo, CA). ..

    Article Title: Organization of the Indian hedgehog – parathyroid hormone-related protein system in the postnatal growth plate
    Article Snippet: .. Reactions were run in triplicate with TaqMan Universal PCR Master Mix (Applied Biosystems) or SYBR Green Master Mix (Applied Biosystems) using the ABI prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. ..

    Article Title: Glutamic acid promotes hair growth in mice
    Article Snippet: .. Gene expression analysis was performed via RT-qPCR using TaqMan Universal PCR Master Mix (7500 detection system, Applied Biosystems). ..

    Article Title: Shedding light on toxicity of SARS-CoV-2 peptides in aquatic biota: A study involving neotropical mosquito larvae (Diptera: Culicidae)
    Article Snippet: .. Briefly, TaqMan® Universal PCR Master Mix (ThermoFisher Scientific, USA) reaction was set up as follows: 10 μL reactions included 2.5 μL of Nuclease-free water, 1.5 μL of Primer and Probe mix, 5 μL of Master Mix and 1 μL of cDNA from the template. ..

    RNA Extraction:

    Article Title: Diverse effects of pan-ROCK and ROCK2 inhibitors on 2 D and 3D cultured human trabecular meshwork (HTM) cells treated with TGFβ2
    Article Snippet: .. Total RNA extraction, reverse transcription and following real-time PCR with the Universal Taqman Master mix using a StepOnePlus instrument (Applied Biosystems/Thermo Fisher Scientific) were performed as describe previously . ..

    Article Title: Hyperosmolarity-Induced Cornification of Human Corneal Epithelial Cells Is Regulated by JNK MAPK
    Article Snippet: .. Alexa Fluor 488–conjugated goat IgG secondary antibodies and fluorescein cadaverine (F-CDV) from Invitrogen-Molecular Probes (Eugene, OR); Hoechst 33342, propidium iodide (PI), and DNA size markers from Sigma-Aldrich; RNA extraction kits (RNeasy Mini Kit, RNAi kit, and siRNA-fluorescein, siRNA-F) from Qiagen (Valencia, CA); a PCR kit (GeneAmp RNA-PCR kit and Taqman Universal PCR master mix; Applied Biosystems, Foster City, CA); a cDNA synthesis kit (Ready-To-Go-Primer First-Strand Beads) from GE Health Care, Inc. (Piscataway, NJ); nuclear and cytoplasmic extraction reagents (NE-PER) and a BCA protein assay kit from Pierce (Rockford, IL); gels for protein electrophoresis (4%–15% Tris-HCl; Ready Gel) from Bio-Rad (Hercules, CA); polyvinylidene difluoride (PVDF; Immobilon-P) membrane from Millipore (Billerica, MA); a chemiluminescent reagent (Luminol) from Santa Cruz Biotechnology (Santa Cruz, CA); an in situ TUNEL kit from Roche (Basel, Switzerland); MTT cell proliferation assay kit from R & D Systems, Inc. (Minneapolis, MN); a JNK cell signaling assay (Beadlyte) and an anti-phosphatidylserine–conjugated Alexa 488 antibody from Upstate Biotechnology (Charlottesville, VA); and an Annexin-V FITC cell viability kit from ApoTarget (Camarillo, CA). ..

    Bicinchoninic Acid Protein Assay:

    Article Title: Hyperosmolarity-Induced Cornification of Human Corneal Epithelial Cells Is Regulated by JNK MAPK
    Article Snippet: .. Alexa Fluor 488–conjugated goat IgG secondary antibodies and fluorescein cadaverine (F-CDV) from Invitrogen-Molecular Probes (Eugene, OR); Hoechst 33342, propidium iodide (PI), and DNA size markers from Sigma-Aldrich; RNA extraction kits (RNeasy Mini Kit, RNAi kit, and siRNA-fluorescein, siRNA-F) from Qiagen (Valencia, CA); a PCR kit (GeneAmp RNA-PCR kit and Taqman Universal PCR master mix; Applied Biosystems, Foster City, CA); a cDNA synthesis kit (Ready-To-Go-Primer First-Strand Beads) from GE Health Care, Inc. (Piscataway, NJ); nuclear and cytoplasmic extraction reagents (NE-PER) and a BCA protein assay kit from Pierce (Rockford, IL); gels for protein electrophoresis (4%–15% Tris-HCl; Ready Gel) from Bio-Rad (Hercules, CA); polyvinylidene difluoride (PVDF; Immobilon-P) membrane from Millipore (Billerica, MA); a chemiluminescent reagent (Luminol) from Santa Cruz Biotechnology (Santa Cruz, CA); an in situ TUNEL kit from Roche (Basel, Switzerland); MTT cell proliferation assay kit from R & D Systems, Inc. (Minneapolis, MN); a JNK cell signaling assay (Beadlyte) and an anti-phosphatidylserine–conjugated Alexa 488 antibody from Upstate Biotechnology (Charlottesville, VA); and an Annexin-V FITC cell viability kit from ApoTarget (Camarillo, CA). ..

    Protein Electrophoresis:

    Article Title: Hyperosmolarity-Induced Cornification of Human Corneal Epithelial Cells Is Regulated by JNK MAPK
    Article Snippet: .. Alexa Fluor 488–conjugated goat IgG secondary antibodies and fluorescein cadaverine (F-CDV) from Invitrogen-Molecular Probes (Eugene, OR); Hoechst 33342, propidium iodide (PI), and DNA size markers from Sigma-Aldrich; RNA extraction kits (RNeasy Mini Kit, RNAi kit, and siRNA-fluorescein, siRNA-F) from Qiagen (Valencia, CA); a PCR kit (GeneAmp RNA-PCR kit and Taqman Universal PCR master mix; Applied Biosystems, Foster City, CA); a cDNA synthesis kit (Ready-To-Go-Primer First-Strand Beads) from GE Health Care, Inc. (Piscataway, NJ); nuclear and cytoplasmic extraction reagents (NE-PER) and a BCA protein assay kit from Pierce (Rockford, IL); gels for protein electrophoresis (4%–15% Tris-HCl; Ready Gel) from Bio-Rad (Hercules, CA); polyvinylidene difluoride (PVDF; Immobilon-P) membrane from Millipore (Billerica, MA); a chemiluminescent reagent (Luminol) from Santa Cruz Biotechnology (Santa Cruz, CA); an in situ TUNEL kit from Roche (Basel, Switzerland); MTT cell proliferation assay kit from R & D Systems, Inc. (Minneapolis, MN); a JNK cell signaling assay (Beadlyte) and an anti-phosphatidylserine–conjugated Alexa 488 antibody from Upstate Biotechnology (Charlottesville, VA); and an Annexin-V FITC cell viability kit from ApoTarget (Camarillo, CA). ..

    In Situ:

    Article Title: Hyperosmolarity-Induced Cornification of Human Corneal Epithelial Cells Is Regulated by JNK MAPK
    Article Snippet: .. Alexa Fluor 488–conjugated goat IgG secondary antibodies and fluorescein cadaverine (F-CDV) from Invitrogen-Molecular Probes (Eugene, OR); Hoechst 33342, propidium iodide (PI), and DNA size markers from Sigma-Aldrich; RNA extraction kits (RNeasy Mini Kit, RNAi kit, and siRNA-fluorescein, siRNA-F) from Qiagen (Valencia, CA); a PCR kit (GeneAmp RNA-PCR kit and Taqman Universal PCR master mix; Applied Biosystems, Foster City, CA); a cDNA synthesis kit (Ready-To-Go-Primer First-Strand Beads) from GE Health Care, Inc. (Piscataway, NJ); nuclear and cytoplasmic extraction reagents (NE-PER) and a BCA protein assay kit from Pierce (Rockford, IL); gels for protein electrophoresis (4%–15% Tris-HCl; Ready Gel) from Bio-Rad (Hercules, CA); polyvinylidene difluoride (PVDF; Immobilon-P) membrane from Millipore (Billerica, MA); a chemiluminescent reagent (Luminol) from Santa Cruz Biotechnology (Santa Cruz, CA); an in situ TUNEL kit from Roche (Basel, Switzerland); MTT cell proliferation assay kit from R & D Systems, Inc. (Minneapolis, MN); a JNK cell signaling assay (Beadlyte) and an anti-phosphatidylserine–conjugated Alexa 488 antibody from Upstate Biotechnology (Charlottesville, VA); and an Annexin-V FITC cell viability kit from ApoTarget (Camarillo, CA). ..

    TUNEL Assay:

    Article Title: Hyperosmolarity-Induced Cornification of Human Corneal Epithelial Cells Is Regulated by JNK MAPK
    Article Snippet: .. Alexa Fluor 488–conjugated goat IgG secondary antibodies and fluorescein cadaverine (F-CDV) from Invitrogen-Molecular Probes (Eugene, OR); Hoechst 33342, propidium iodide (PI), and DNA size markers from Sigma-Aldrich; RNA extraction kits (RNeasy Mini Kit, RNAi kit, and siRNA-fluorescein, siRNA-F) from Qiagen (Valencia, CA); a PCR kit (GeneAmp RNA-PCR kit and Taqman Universal PCR master mix; Applied Biosystems, Foster City, CA); a cDNA synthesis kit (Ready-To-Go-Primer First-Strand Beads) from GE Health Care, Inc. (Piscataway, NJ); nuclear and cytoplasmic extraction reagents (NE-PER) and a BCA protein assay kit from Pierce (Rockford, IL); gels for protein electrophoresis (4%–15% Tris-HCl; Ready Gel) from Bio-Rad (Hercules, CA); polyvinylidene difluoride (PVDF; Immobilon-P) membrane from Millipore (Billerica, MA); a chemiluminescent reagent (Luminol) from Santa Cruz Biotechnology (Santa Cruz, CA); an in situ TUNEL kit from Roche (Basel, Switzerland); MTT cell proliferation assay kit from R & D Systems, Inc. (Minneapolis, MN); a JNK cell signaling assay (Beadlyte) and an anti-phosphatidylserine–conjugated Alexa 488 antibody from Upstate Biotechnology (Charlottesville, VA); and an Annexin-V FITC cell viability kit from ApoTarget (Camarillo, CA). ..

    MTT Assay:

    Article Title: Hyperosmolarity-Induced Cornification of Human Corneal Epithelial Cells Is Regulated by JNK MAPK
    Article Snippet: .. Alexa Fluor 488–conjugated goat IgG secondary antibodies and fluorescein cadaverine (F-CDV) from Invitrogen-Molecular Probes (Eugene, OR); Hoechst 33342, propidium iodide (PI), and DNA size markers from Sigma-Aldrich; RNA extraction kits (RNeasy Mini Kit, RNAi kit, and siRNA-fluorescein, siRNA-F) from Qiagen (Valencia, CA); a PCR kit (GeneAmp RNA-PCR kit and Taqman Universal PCR master mix; Applied Biosystems, Foster City, CA); a cDNA synthesis kit (Ready-To-Go-Primer First-Strand Beads) from GE Health Care, Inc. (Piscataway, NJ); nuclear and cytoplasmic extraction reagents (NE-PER) and a BCA protein assay kit from Pierce (Rockford, IL); gels for protein electrophoresis (4%–15% Tris-HCl; Ready Gel) from Bio-Rad (Hercules, CA); polyvinylidene difluoride (PVDF; Immobilon-P) membrane from Millipore (Billerica, MA); a chemiluminescent reagent (Luminol) from Santa Cruz Biotechnology (Santa Cruz, CA); an in situ TUNEL kit from Roche (Basel, Switzerland); MTT cell proliferation assay kit from R & D Systems, Inc. (Minneapolis, MN); a JNK cell signaling assay (Beadlyte) and an anti-phosphatidylserine–conjugated Alexa 488 antibody from Upstate Biotechnology (Charlottesville, VA); and an Annexin-V FITC cell viability kit from ApoTarget (Camarillo, CA). ..

    Proliferation Assay:

    Article Title: Hyperosmolarity-Induced Cornification of Human Corneal Epithelial Cells Is Regulated by JNK MAPK
    Article Snippet: .. Alexa Fluor 488–conjugated goat IgG secondary antibodies and fluorescein cadaverine (F-CDV) from Invitrogen-Molecular Probes (Eugene, OR); Hoechst 33342, propidium iodide (PI), and DNA size markers from Sigma-Aldrich; RNA extraction kits (RNeasy Mini Kit, RNAi kit, and siRNA-fluorescein, siRNA-F) from Qiagen (Valencia, CA); a PCR kit (GeneAmp RNA-PCR kit and Taqman Universal PCR master mix; Applied Biosystems, Foster City, CA); a cDNA synthesis kit (Ready-To-Go-Primer First-Strand Beads) from GE Health Care, Inc. (Piscataway, NJ); nuclear and cytoplasmic extraction reagents (NE-PER) and a BCA protein assay kit from Pierce (Rockford, IL); gels for protein electrophoresis (4%–15% Tris-HCl; Ready Gel) from Bio-Rad (Hercules, CA); polyvinylidene difluoride (PVDF; Immobilon-P) membrane from Millipore (Billerica, MA); a chemiluminescent reagent (Luminol) from Santa Cruz Biotechnology (Santa Cruz, CA); an in situ TUNEL kit from Roche (Basel, Switzerland); MTT cell proliferation assay kit from R & D Systems, Inc. (Minneapolis, MN); a JNK cell signaling assay (Beadlyte) and an anti-phosphatidylserine–conjugated Alexa 488 antibody from Upstate Biotechnology (Charlottesville, VA); and an Annexin-V FITC cell viability kit from ApoTarget (Camarillo, CA). ..

    SYBR Green Assay:

    Article Title: Organization of the Indian hedgehog – parathyroid hormone-related protein system in the postnatal growth plate
    Article Snippet: .. Reactions were run in triplicate with TaqMan Universal PCR Master Mix (Applied Biosystems) or SYBR Green Master Mix (Applied Biosystems) using the ABI prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. ..

    Sequencing:

    Article Title: Organization of the Indian hedgehog – parathyroid hormone-related protein system in the postnatal growth plate
    Article Snippet: .. Reactions were run in triplicate with TaqMan Universal PCR Master Mix (Applied Biosystems) or SYBR Green Master Mix (Applied Biosystems) using the ABI prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. ..

    Expressing:

    Article Title: Glutamic acid promotes hair growth in mice
    Article Snippet: .. Gene expression analysis was performed via RT-qPCR using TaqMan Universal PCR Master Mix (7500 detection system, Applied Biosystems). ..

    Quantitative RT-PCR:

    Article Title: Glutamic acid promotes hair growth in mice
    Article Snippet: .. Gene expression analysis was performed via RT-qPCR using TaqMan Universal PCR Master Mix (7500 detection system, Applied Biosystems). ..

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    Thermo Fisher taqman universal pcr master mix
    MiRNA validation. Nineteen miRNAs from our deep sequencing data were selected for further validation using real-time <t>TaqMan®</t> MicroRNA Assays, including nine novel, cat-specific miRNAs. miR-151 and miR-361 were expressed at very consistent levels in all of the deep sequencing samples (not shown) and, thus, <t>qRT-PCR</t> values were normalized to these two miRs. Figure shows heat maps of averaged and normalized miR counts from the deep sequencing data and of the normalized relative expression from qRT-PCR. All of the normalized deep sequencing and qRT-PCR values for each individual miR in each individual sample are given in Supplementary Table S1.10 .
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    MiRNA validation. Nineteen miRNAs from our deep sequencing data were selected for further validation using real-time TaqMan® MicroRNA Assays, including nine novel, cat-specific miRNAs. miR-151 and miR-361 were expressed at very consistent levels in all of the deep sequencing samples (not shown) and, thus, qRT-PCR values were normalized to these two miRs. Figure shows heat maps of averaged and normalized miR counts from the deep sequencing data and of the normalized relative expression from qRT-PCR. All of the normalized deep sequencing and qRT-PCR values for each individual miR in each individual sample are given in Supplementary Table S1.10 .

    Journal: Scientific Reports

    Article Title: Discovery and characterization of the feline miRNAome

    doi: 10.1038/s41598-017-10164-w

    Figure Lengend Snippet: MiRNA validation. Nineteen miRNAs from our deep sequencing data were selected for further validation using real-time TaqMan® MicroRNA Assays, including nine novel, cat-specific miRNAs. miR-151 and miR-361 were expressed at very consistent levels in all of the deep sequencing samples (not shown) and, thus, qRT-PCR values were normalized to these two miRs. Figure shows heat maps of averaged and normalized miR counts from the deep sequencing data and of the normalized relative expression from qRT-PCR. All of the normalized deep sequencing and qRT-PCR values for each individual miR in each individual sample are given in Supplementary Table S1.10 .

    Article Snippet: The PCR reactions for each sample were performed in duplicate using TaqMan® Universal PCR Master Mix, no AmpErase® UNG (Life Technologies) and the reactions were run in a LightCycler480 (Roche) under the following PCR conditions: 10 min for 95 °C, and 40 cycles of 95 °C for 15 sec and 60 °C for 60 sec. All of the qRT-PCR data was normalized against the 2 miRNAs (miR-151 & 361) that had the lowest combined S.D. (S.D./Average normalized miR counts) in our deep-sequencing data across all 27 tissues, as recommended by Schwarzenbach et al .

    Techniques: Sequencing, Quantitative RT-PCR, Expressing

    Collagen production by isolated cardiac fibroblasts Quantitative Taqman PCR for ColIα1 mRNA from isolated cardiac fibroblasts treated with A. TGF-β1 (20ng/ml), n=4 B. conditioned media from nontransgenic (NTG) or SR-uPA +/0 macrophages, n=4 each group, C. plasmin (5nM), n=7, D. conditioned media from nontransgenic (NTG) macrophages treated with M199 alone or M199 with plasmin (5nM), n=9. Data represent mean ± S.D. of fibroblasts from individual mice treated with macrophages from different individual mice.

    Journal: Journal of molecular and cellular cardiology

    Article Title: The Role of Macrophage Derived Urokinase Plasminogen Activator in Myocardial Infarct Repair

    doi: 10.1016/j.yjmcc.2010.03.022

    Figure Lengend Snippet: Collagen production by isolated cardiac fibroblasts Quantitative Taqman PCR for ColIα1 mRNA from isolated cardiac fibroblasts treated with A. TGF-β1 (20ng/ml), n=4 B. conditioned media from nontransgenic (NTG) or SR-uPA +/0 macrophages, n=4 each group, C. plasmin (5nM), n=7, D. conditioned media from nontransgenic (NTG) macrophages treated with M199 alone or M199 with plasmin (5nM), n=9. Data represent mean ± S.D. of fibroblasts from individual mice treated with macrophages from different individual mice.

    Article Snippet: Primer/probe sets for ColIα1 (Mm00801666_g1) were pre-validated sets obtained from Applied Biosystems (Inventoried TaqMan® Gene Expression Assays: cat# 4331182). qPCR was performed using the Taqman® Universal PCR Mastermix from Applied Biosystems (cat# 4304437).

    Techniques: Isolation, Polymerase Chain Reaction, Mouse Assay

    Expression of uPA and collagen content in M29 SR-uPA +/0 mice A. Quantitative Taqman PCR for uPA mRNA in isolated peritoneal macrophages from nontransgenic, SR-uPA M29 and SR-uPA M87 mice. B. Percent fibrillar collagen in uninfarcted nontransgenic and SR-uPA M29 hearts. Picrosirius red stain of uninfarcted hearts at 3 months of age from C. nontransgenic, and D. SR-uPA M29 mice.

    Journal: Journal of molecular and cellular cardiology

    Article Title: The Role of Macrophage Derived Urokinase Plasminogen Activator in Myocardial Infarct Repair

    doi: 10.1016/j.yjmcc.2010.03.022

    Figure Lengend Snippet: Expression of uPA and collagen content in M29 SR-uPA +/0 mice A. Quantitative Taqman PCR for uPA mRNA in isolated peritoneal macrophages from nontransgenic, SR-uPA M29 and SR-uPA M87 mice. B. Percent fibrillar collagen in uninfarcted nontransgenic and SR-uPA M29 hearts. Picrosirius red stain of uninfarcted hearts at 3 months of age from C. nontransgenic, and D. SR-uPA M29 mice.

    Article Snippet: Primer/probe sets for ColIα1 (Mm00801666_g1) were pre-validated sets obtained from Applied Biosystems (Inventoried TaqMan® Gene Expression Assays: cat# 4331182). qPCR was performed using the Taqman® Universal PCR Mastermix from Applied Biosystems (cat# 4304437).

    Techniques: Expressing, Mouse Assay, Polymerase Chain Reaction, Isolation, Staining

    Lenalidomide modulates cytokines and IGF-1 profiles in myeloma cells. ( A ) Expression of TNF- α mRNA in myeloma cells and BMSC. ( B ) Modulation of TNF- α mRNA induced by LEN treatment in myeloma cells and BMSC. Human myeloma cell lines, primary CD138+ MM cells and BMSCs were incubated with or without 10 μ ℳ LEN for 24 h. Total RNA was extracted and reverse-transcribed as described previously. Tumour necrosis factor- α mRNA levels were evaluated by real-time PCR using the TaqMan probe Hs00174128-m1 (Applied Biosystem). The relative expression of TNF- α mRNA was calculated according to the equation of Pfaffl and normalised to LP1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate. ( C ) Increase of TNF- α production by LEN in myeloma cell lines. ( D ) Expression of IGF-1 mRNA in myeloma cells and BMSCs. ( E ) Increase in IGF-1 mRNA levels under LEN treatment. Insulin-like growth factor-1 mRNA levels were evaluated as above using the TaqMan gene expression assay Hs 00153126-m1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate.

    Journal: British Journal of Cancer

    Article Title: Paradoxical effect of lenalidomide on cytokine/growth factor profiles in multiple myeloma

    doi: 10.1038/bjc.2013.186

    Figure Lengend Snippet: Lenalidomide modulates cytokines and IGF-1 profiles in myeloma cells. ( A ) Expression of TNF- α mRNA in myeloma cells and BMSC. ( B ) Modulation of TNF- α mRNA induced by LEN treatment in myeloma cells and BMSC. Human myeloma cell lines, primary CD138+ MM cells and BMSCs were incubated with or without 10 μ ℳ LEN for 24 h. Total RNA was extracted and reverse-transcribed as described previously. Tumour necrosis factor- α mRNA levels were evaluated by real-time PCR using the TaqMan probe Hs00174128-m1 (Applied Biosystem). The relative expression of TNF- α mRNA was calculated according to the equation of Pfaffl and normalised to LP1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate. ( C ) Increase of TNF- α production by LEN in myeloma cell lines. ( D ) Expression of IGF-1 mRNA in myeloma cells and BMSCs. ( E ) Increase in IGF-1 mRNA levels under LEN treatment. Insulin-like growth factor-1 mRNA levels were evaluated as above using the TaqMan gene expression assay Hs 00153126-m1. Graphs represent the mean±s.d. of mRNA levels from two independent experiments performed in duplicate.

    Article Snippet: Quantitative PCR was performed in duplicate using the TaqMan Universal PCR Master Mix (Applied Biosystems, Courtaboeuf, France) and the MX3005 instrument (Stratagene, Amsterdam, The Neterlands).

    Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction

    hPSC-Derived MesoT Displays Molecular Characteristics of Primary Mesothelium (A) Sources of cells used for RNA-seq analysis. hPSC-derived MesoT cells and tdTomato+ mesothelium isolated from mouse embryonic gut, liver, heart, and lungs (embryonic day 15.5 [E15.5]) were compared to RNA-seq data in the public domain. (B) Addition of Wnt3a, BMP4, and retinoic acid (RA) to SplM (ISL1 + , NKX2.5 + , ZO1 + ) efficiently generates MesoT (WT1 + , αSMA + , VIM + ) with a mesenchymal phenotype. Scale bars, 50 μm. (C) qRT-PCR data showing fold-change of transcript levels for markers of SplM ( ISL1, NKX2.5, and GATA4 ) and MesoT ( WT1, TBX18, and TCF21 ) following directed differentiation of human embryonic stem cells (hESCs, WA09). TaqMan assays for each transcript were performed in technical triplicate and fold-change shown relative to untreated hESCs (WA09) after normalization with 18S RNA. .

    Journal: Cell reports

    Article Title: Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair

    doi: 10.1016/j.celrep.2019.02.016

    Figure Lengend Snippet: hPSC-Derived MesoT Displays Molecular Characteristics of Primary Mesothelium (A) Sources of cells used for RNA-seq analysis. hPSC-derived MesoT cells and tdTomato+ mesothelium isolated from mouse embryonic gut, liver, heart, and lungs (embryonic day 15.5 [E15.5]) were compared to RNA-seq data in the public domain. (B) Addition of Wnt3a, BMP4, and retinoic acid (RA) to SplM (ISL1 + , NKX2.5 + , ZO1 + ) efficiently generates MesoT (WT1 + , αSMA + , VIM + ) with a mesenchymal phenotype. Scale bars, 50 μm. (C) qRT-PCR data showing fold-change of transcript levels for markers of SplM ( ISL1, NKX2.5, and GATA4 ) and MesoT ( WT1, TBX18, and TCF21 ) following directed differentiation of human embryonic stem cells (hESCs, WA09). TaqMan assays for each transcript were performed in technical triplicate and fold-change shown relative to untreated hESCs (WA09) after normalization with 18S RNA. .

    Article Snippet: mRNA was isolated using the E.Z.N.A.® Total RNA Kit I (Omega Bio-Tek, R6834) followed by quantification using a Biotek Synergy 2 plate reader. cDNA was made using iSCRIPT cDNA kit (Bio-Rad, 1708841). qRT-PCR was performed using TaqMan Universal PCR Master Mix No AmpErase UNG (Thermo Fisher, 4324020) and TaqMan Primers on a ViiA7 Real-Time PCR System (Life Technologies).

    Techniques: Derivative Assay, RNA Sequencing Assay, Isolation, Quantitative RT-PCR