taqman probes Search Results


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  • 99
    Thermo Fisher taqman mgb probes
    Phylogenetic analysis of nucleotide sequences from NDV strains based on a 325-bp-long fragment amplified from the fusion protein genes, which encompasses the annealing sites of oligonucleotide primers and <t>TaqMan</t> <t>MGB</t> probes described in the RRT-PCR pathotyping
    Taqman Mgb Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher taqman probes
    Expression of COX4/1 and PGC1α in SK-N-SH cells and HepG2 cells after incubation of the cells with PQQ, PQQH 2 and IPQ. (A) COX4/1 expression in SK-N-SH cells, (B) COX4/1 expression in HepG2 cells (C) PGC1α expression in SK-N-SH cells, (D) PGC1α expression in HepG2 cells. Total mRNAs were extracted from SK-N-SH cells and HepG2 cells that had been cultured for 24 h after the addition PQQ, PQQH 2 and IPQ. One hundred nM and 1 μM of PQQ, PQQH 2 or IPQ were added to SK-N-SH cells and HepG2 cells, respectively. Total RNAs were isolated from cultured cells. Total RNAs (3–5 μg) were transcribed into cDNA by real-time quantitative polymerase chain reactions with <t>TaqMan</t> probes for COX4/1 and PGC1α. The relative quantities of COX4/1 and PGC1α transcripts were normalized to the level of GAPDH message. Each value is the mean ± SEM (n = 3–5). ***p
    Taqman Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 31361 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taqman gene expression assay probes
    Expression of COX4/1 and PGC1α in SK-N-SH cells and HepG2 cells after incubation of the cells with PQQ, PQQH 2 and IPQ. (A) COX4/1 expression in SK-N-SH cells, (B) COX4/1 expression in HepG2 cells (C) PGC1α expression in SK-N-SH cells, (D) PGC1α expression in HepG2 cells. Total mRNAs were extracted from SK-N-SH cells and HepG2 cells that had been cultured for 24 h after the addition PQQ, PQQH 2 and IPQ. One hundred nM and 1 μM of PQQ, PQQH 2 or IPQ were added to SK-N-SH cells and HepG2 cells, respectively. Total RNAs were isolated from cultured cells. Total RNAs (3–5 μg) were transcribed into cDNA by real-time quantitative polymerase chain reactions with <t>TaqMan</t> probes for COX4/1 and PGC1α. The relative quantities of COX4/1 and PGC1α transcripts were normalized to the level of GAPDH message. Each value is the mean ± SEM (n = 3–5). ***p
    Taqman Gene Expression Assay Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 804 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taqman gene expression probes
    Hypoxia-induced modification of PLK promoter methylation in HCC cells. (a) Promoter methylation status of the plks examined in HCC-derived cells HepG2 and Hep3B; U = unmethylated, M = methylated. Fully methylated HeLa DNA was used as a positive control (+M), no template was added to the negative control (−M). (b) Post hypoxia, PLK4 transcripts were assessed via qPCR in RNA extracted from HCC cells. All qPCR data is representative of the mean value of three independent experiments and error bars represent +/− SD. (c) PLK protein levels were examined post treatment from whole cell lysates. Actin was used as a loading control. (−) represents lysates from untreated cells, (+) lysates from cells grown in the presence of hypoxia. (d) Quantification of protein levels using densitometry. Levels have been normalized to the respective untreated controls. Data is representative of the mean value of three independent experiments and error bars represent +/− SD. (e) The fold change of <t>PLK1</t> transcripts as determined by qPCR. Values normalized to the respective untreated sample. (f) PLK2 and PLK3 analyzed and fold changed determine by normalization to the respective untreated samples. (g) Hif1α transcripts post hypoxia were determine by real-time PCR using a <t>Taqman</t> probe.
    Taqman Gene Expression Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taqman mirna probes
    Downregulation of expression levels of miR-17, miR-20a, and miR-106a in promyelocytic cells during SIRPα protein induction by cell differentiation agents. A–C, Induction of SIRPα protein but not SIRPα mRNA by TPA (30 nmol/L) in promyelocytic HL-60 and U937 cells. Note that the SIRPα protein level (Fig 1, A , upper panel , and B ) was significantly increased by TPA, whereas the SIRPα mRNA level (Fig 1, A , lower panel , and C ) was not altered. D, Microarray analysis of changes in <t>miRNA</t> expression in TPA-differentiated HL-60 cells. E, <t>TaqMan</t> probe–based quantitative RT-PCR validation of differentially expressed miRNAs. F and G, Inverse correlation between SIRPα protein levels (Fig 1, F ) and levels of miR-17/20a/106a (Fig 1, G ) in HL-60 cells during RA-induced differentiation process. Data represent means ± SDs of 3 independent experiments performed in triplicate. * P
    Taqman Mirna Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1048 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher gene specific taqman probes
    Periostin increases in gingival healing following gingivectomy. ( A ) Gingival tissues dissected from rats were assessed for POSTN gene expression. Isoform analysis using <t>RT-PCR</t> demonstrated the presence of 2 isoforms of POSTN . ( B ) Analysis of the wild-type isoform of POSTN using <t>Taqman</t> PCR showed periostin mRNA peaked at day 7 post wounding. Data represents mean fold gene expressions ± s.d. relative to control day 0 of 3 independent rats. Data was analyzed via one-way ANOVA (* p
    Gene Specific Taqman Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taqman assay probes
    High-density SNP mapping in the HBB locus. (a) Distribution of 103 <t>SNPs</t> in the HBB locus interrogated by the Illumina Omni1-Quad chip and <t>TaqMan</t> assay. Globin genes are indicated by boxes. The schematic is not drawn to scale. Abbreviations: LCR, locus
    Taqman Assay Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad taqman probes
    Comparison of multiplex and <t>singleplex</t> assays with <t>TaqMan</t> probe‐based detection. (a) multiplex amplification with mixed TaqMan oligos, mixed TaqMan probes, and mixed target DNA as template. (b–f) Singleplex TaqMan amplification signals when species‐specific TaqMan oligos, species‐specific TaqMan probes, and specific target DNA were used (lines with markers) relative to multiplex amplification signal when mixed TaqMan oligos, mixed TaqMan probes, and mixed target DNA were used (straight lines)
    Taqman Probes, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 494 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher fam labeled taqman probes
    TTP regulation of CXCR4 mRNA and protein. ( A ) Quantitative PCR (qPCR) quantification of CXCR4 mRNA associated with TTP protein. MDA-MB-231 cells were transfected with TTP or C124R expression plasmids for 24h. Cells were lysed, and TTP and C124R proteins were immunoprecipitated using anti-TTP or normal IgG control antibody. Quantification of associated CXCR4 mRNA was performed by qPCR using a <t>FAM-labeled</t> human CXCR4 <t>Taqman</t> expression probe and normalized to a VIC-labeled GAPDH probe. Data are from one experiment representative of two independent experiments * P
    Fam Labeled Taqman Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taqman tamra probe 5 vic
    TTP regulation of CXCR4 mRNA and protein. ( A ) Quantitative PCR (qPCR) quantification of CXCR4 mRNA associated with TTP protein. MDA-MB-231 cells were transfected with TTP or C124R expression plasmids for 24h. Cells were lysed, and TTP and C124R proteins were immunoprecipitated using anti-TTP or normal IgG control antibody. Quantification of associated CXCR4 mRNA was performed by qPCR using a <t>FAM-labeled</t> human CXCR4 <t>Taqman</t> expression probe and normalized to a VIC-labeled GAPDH probe. Data are from one experiment representative of two independent experiments * P
    Taqman Tamra Probe 5 Vic, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher custom taqman probes
    Accumulation of spontaneous tumorigenic Kras mutations in Sirt2 -deficient mice. (a) Accumulation of tumorigenic Kras mutations during inflammation-coupled neoplasm. Mouse pancreas genomic <t>DNA</t> from wild-type and Sirt2 KO, with and without caerulein-treatment, were analyzed for Kras G12D or Kras G12V mutations by competitive allele-specific <t>TaqMan</t> PCR. The table shows the summary of analysis for percentage of mice positive for mutations (46 wild-type mice, 56 Sirt2 KO mice) after pancreatitis. (b) Detail description of wild-type and Sirt2 KO mice with Kras G12D and Kras G12V mutations. (c) Bar graph representation of ( b ). ( d ) Immunostaining detection of KRAS-G12D mutant protein in mouse pancreas. Mice pancreas tissues were fixed, embedded in paraffin, and immuno-histochemical staining with anti- KRAS-G12D antibody was performed. Representative images are shown. Bars indicate 100 µm.
    Custom Taqman Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher taqman tamra probe
    Accumulation of spontaneous tumorigenic Kras mutations in Sirt2 -deficient mice. (a) Accumulation of tumorigenic Kras mutations during inflammation-coupled neoplasm. Mouse pancreas genomic <t>DNA</t> from wild-type and Sirt2 KO, with and without caerulein-treatment, were analyzed for Kras G12D or Kras G12V mutations by competitive allele-specific <t>TaqMan</t> PCR. The table shows the summary of analysis for percentage of mice positive for mutations (46 wild-type mice, 56 Sirt2 KO mice) after pancreatitis. (b) Detail description of wild-type and Sirt2 KO mice with Kras G12D and Kras G12V mutations. (c) Bar graph representation of ( b ). ( d ) Immunostaining detection of KRAS-G12D mutant protein in mouse pancreas. Mice pancreas tissues were fixed, embedded in paraffin, and immuno-histochemical staining with anti- KRAS-G12D antibody was performed. Representative images are shown. Bars indicate 100 µm.
    Taqman Tamra Probe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Kaneka Corp taqman probes
    (a) Schematic representation of the agr operon organisation and regulation in Staphylococcus aureus . (b) Schematic representation showing single-site chromosomal integration of GFP transcriptional fusion reporters for P2 and P3. (c) Sequence of P2 and P3 promoter regions used to generate the gfp transcriptional fusions for P2 and P3 (the consensus −10 and −35 sites are outlined, the transcription start sites are shown with arrows and the RBS is underlined. (d) Sheep blood agar haemolysis assay with S. aureus USA300 WT (1), USA300 agrA ::Tn [ agrA transposon mutant from NARSA library (Fey et al ., 2013 )] (2), USA300 agr IR P3-GFP (3) and USA300 agr IR P2-GFP (4). (e) Graphs showing relative hld , agrA and gyrB mRNA levels in 16 h cultures of S. aureus USA300 WT and USA300 agr IR P3-GFP as determined by <t>Taqman</t> <t>qRT-PCR.</t> Values are shown for each gene with respect to USA300 WT levels. (f) Graph showing growth curves (OD 600 ) of S. aureus USA300 WT , USA300 agr IR P2-GFP and USA300 agr IR P3-GFP strains grown in TSB. (g) Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] and GFP-FU as a function of growth (OD 600 ) over time for S. aureus USA300 agr IR P2-GFP and USA300 agr IR P3-GFP strains grown in TSB. (h) As in (g) but with S. aureus SH1000 agr IR P2-GFP , SH1000 agr IR P3-GFP , SH1001 agr IR P2-GFP and SH1001 agr IR P3-GFP strains grown in TSB. Data for (d–h) were obtained from three biological replicates
    Taqman Probes, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 92/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Phylogenetic analysis of nucleotide sequences from NDV strains based on a 325-bp-long fragment amplified from the fusion protein genes, which encompasses the annealing sites of oligonucleotide primers and TaqMan MGB probes described in the RRT-PCR pathotyping

    Journal: Journal of Clinical Microbiology

    Article Title: Real-Time PCR-Based Pathotyping of Newcastle Disease Virus by Use of TaqMan Minor Groove Binder Probes ▿

    doi: 10.1128/JCM.01652-08

    Figure Lengend Snippet: Phylogenetic analysis of nucleotide sequences from NDV strains based on a 325-bp-long fragment amplified from the fusion protein genes, which encompasses the annealing sites of oligonucleotide primers and TaqMan MGB probes described in the RRT-PCR pathotyping

    Article Snippet: For real-time PCR amplification, the cycling program recommended by the manufacturer of the TaqMan MGB probes (Applied Biosystems) was used: 95°C for 10 min (hot start), followed by 45 cycles of 95°C for 15 s and 60°C for 60 s. Fluorescence data were collected in the primer elongation phase at 60°C.

    Techniques: Amplification, Quantitative RT-PCR

    Amplification plots of Ceratocystis platani DNA showing comparison between two different TaqMan MGB probes. The fluorescence (normalized reporter signal, Rn) of the TaqMan MGB probes for the CP target gene (broken line) appears later than the fluorescence

    Journal: Applied and Environmental Microbiology

    Article Title: Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

    doi: 10.1128/AEM.01484-13

    Figure Lengend Snippet: Amplification plots of Ceratocystis platani DNA showing comparison between two different TaqMan MGB probes. The fluorescence (normalized reporter signal, Rn) of the TaqMan MGB probes for the CP target gene (broken line) appears later than the fluorescence

    Article Snippet: The primer and probe final concentrations used were as follows: 300 nM forward primer (Eurofins MWG Operon, Ebersberg, Germany), 300 nM reverse primer (Eurofins MWG Operon), and 200 nM TaqMan MGB probe (Applied Biosystems) designed for amplification of ITS and CP genes.

    Techniques: Amplification, Fluorescence

    TaqMan MGB probes and primer specificity.

    Journal: Applied and Environmental Microbiology

    Article Title: Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

    doi: 10.1128/AEM.01484-13

    Figure Lengend Snippet: TaqMan MGB probes and primer specificity.

    Article Snippet: The primer and probe final concentrations used were as follows: 300 nM forward primer (Eurofins MWG Operon, Ebersberg, Germany), 300 nM reverse primer (Eurofins MWG Operon), and 200 nM TaqMan MGB probe (Applied Biosystems) designed for amplification of ITS and CP genes.

    Techniques:

    Ceratocystis platani detection by TaqMan MGB probe on ITS2. Selection of amplification plots from different DNA samples: (i) C. platani mycelium and infected plane tissues (continuous lines), (ii) airborne inoculum traps (AITs) from sampling area (broken

    Journal: Applied and Environmental Microbiology

    Article Title: Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

    doi: 10.1128/AEM.01484-13

    Figure Lengend Snippet: Ceratocystis platani detection by TaqMan MGB probe on ITS2. Selection of amplification plots from different DNA samples: (i) C. platani mycelium and infected plane tissues (continuous lines), (ii) airborne inoculum traps (AITs) from sampling area (broken

    Article Snippet: The primer and probe final concentrations used were as follows: 300 nM forward primer (Eurofins MWG Operon, Ebersberg, Germany), 300 nM reverse primer (Eurofins MWG Operon), and 200 nM TaqMan MGB probe (Applied Biosystems) designed for amplification of ITS and CP genes.

    Techniques: Selection, Amplification, Infection, Sampling

    Alignment of two primers and the TaqMan MGB probe sequences against the internal transcribed spacer (ITS) and cerato-platanin (CP) gene for which the assay was designed. Primer sequences are indicated by the arrow boxes, and the probe sequences are shown

    Journal: Applied and Environmental Microbiology

    Article Title: Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

    doi: 10.1128/AEM.01484-13

    Figure Lengend Snippet: Alignment of two primers and the TaqMan MGB probe sequences against the internal transcribed spacer (ITS) and cerato-platanin (CP) gene for which the assay was designed. Primer sequences are indicated by the arrow boxes, and the probe sequences are shown

    Article Snippet: The primer and probe final concentrations used were as follows: 300 nM forward primer (Eurofins MWG Operon, Ebersberg, Germany), 300 nM reverse primer (Eurofins MWG Operon), and 200 nM TaqMan MGB probe (Applied Biosystems) designed for amplification of ITS and CP genes.

    Techniques:

    TaqMan MGB probes and primer specificity.

    Journal: Applied and Environmental Microbiology

    Article Title: Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

    doi: 10.1128/AEM.01484-13

    Figure Lengend Snippet: TaqMan MGB probes and primer specificity.

    Article Snippet: The primer and probe final concentrations used were as follows: 300 nM forward primer (Eurofins MWG Operon, Ebersberg, Germany), 300 nM reverse primer (Eurofins MWG Operon), and 200 nM TaqMan MGB probe (Applied Biosystems) designed for amplification of ITS and CP genes.

    Techniques:

    Expression of COX4/1 and PGC1α in SK-N-SH cells and HepG2 cells after incubation of the cells with PQQ, PQQH 2 and IPQ. (A) COX4/1 expression in SK-N-SH cells, (B) COX4/1 expression in HepG2 cells (C) PGC1α expression in SK-N-SH cells, (D) PGC1α expression in HepG2 cells. Total mRNAs were extracted from SK-N-SH cells and HepG2 cells that had been cultured for 24 h after the addition PQQ, PQQH 2 and IPQ. One hundred nM and 1 μM of PQQ, PQQH 2 or IPQ were added to SK-N-SH cells and HepG2 cells, respectively. Total RNAs were isolated from cultured cells. Total RNAs (3–5 μg) were transcribed into cDNA by real-time quantitative polymerase chain reactions with TaqMan probes for COX4/1 and PGC1α. The relative quantities of COX4/1 and PGC1α transcripts were normalized to the level of GAPDH message. Each value is the mean ± SEM (n = 3–5). ***p

    Journal: Heliyon

    Article Title: Effects of pyrroloquinoline quinone and imidazole pyrroloquinoline on biological activities and neural functions

    doi: 10.1016/j.heliyon.2020.e03240

    Figure Lengend Snippet: Expression of COX4/1 and PGC1α in SK-N-SH cells and HepG2 cells after incubation of the cells with PQQ, PQQH 2 and IPQ. (A) COX4/1 expression in SK-N-SH cells, (B) COX4/1 expression in HepG2 cells (C) PGC1α expression in SK-N-SH cells, (D) PGC1α expression in HepG2 cells. Total mRNAs were extracted from SK-N-SH cells and HepG2 cells that had been cultured for 24 h after the addition PQQ, PQQH 2 and IPQ. One hundred nM and 1 μM of PQQ, PQQH 2 or IPQ were added to SK-N-SH cells and HepG2 cells, respectively. Total RNAs were isolated from cultured cells. Total RNAs (3–5 μg) were transcribed into cDNA by real-time quantitative polymerase chain reactions with TaqMan probes for COX4/1 and PGC1α. The relative quantities of COX4/1 and PGC1α transcripts were normalized to the level of GAPDH message. Each value is the mean ± SEM (n = 3–5). ***p

    Article Snippet: Real-time quantitative polymerase chain reactions were performed in an Eco real-time PCR system (Illumia, San Diego, CA) in combination with TaqMan probes (Applied Biosystems) for COX4/1 and PGC1 α.

    Techniques: Expressing, Incubation, Cell Culture, Isolation

    RECQL5 mRNA is increased in UCC and is associated with poor prognosis Taqman qRT-PCR quantification of mRNA from 197 primary bladder tumour and 20 normal tissue samples using a RECQL5 probe and plotted as fold change [ 52 ]. A. Stratified by normal compared to malignant tissue and B. according to tumour grade; * and *** indicate p

    Journal: Oncotarget

    Article Title: Altered RECQL5 expression in urothelial bladder carcinoma increases cellular proliferation and makes RECQL5 helicase activity a novel target for chemotherapy

    doi: 10.18632/oncotarget.12683

    Figure Lengend Snippet: RECQL5 mRNA is increased in UCC and is associated with poor prognosis Taqman qRT-PCR quantification of mRNA from 197 primary bladder tumour and 20 normal tissue samples using a RECQL5 probe and plotted as fold change [ 52 ]. A. Stratified by normal compared to malignant tissue and B. according to tumour grade; * and *** indicate p

    Article Snippet: RT-PCR was performed using TaqMan probe and primers (Applied Biosystems, Grand Island, NY) for RECQL5β (Hs00696986_g1) and RECQL5 non isotype specific (Hs00188633_m1), Heat shock protein 90kDa alpha (cytosolic), class B member 1 (HSP90AB1) (Hs03043878_g1), Testis enhanced gene transcript protein (TEGT) (Hs01012085_m1) and Mitochondrial ATP synthase H+ transporting F1 complex beta subunit (ATP5B) (Hs00969573_mH) with TaqMan Universal PCR Mix (Applied Biosystems) as recommended by the manufacturer.

    Techniques: Quantitative RT-PCR

    Confirmation of Lpcat1 GT in ES cells and generation of mice. ( A ) Top: Diagram of Lpcat1 locus. Boxes denote exons, and intervening lines denote introns. The β-geo selection cassette encoding a β-galactosidase/neomycin fusion protein was inserted in intron 9, approximately 400 bp 3′ of exon 9. f1, f2, and r2 denote primers used to confirm the presence of GT cassette in the Lpcat1 locus by qPCR analysis. Bottom: PCR analysis of the Lpcat1 locus in GT ES cells or parental ES cells (129J/Ola) using 2 separate primer sets. The presence of amplicons in GT ES cells with the absence of signal in parental cells demonstrates trapping of the Lpcat1 locus at exon 9. Vertical line indicates discontinuous lanes in the same gel. ( B ) qPCR analysis of GT ES cells. cDNA from GT ES cells (GT) and parental cells (WT) was subjected to qPCR analysis using 3 separate exon-spanning TaqMan primer/probe sets for Lpcat1 as depicted in the bottom panel: 2/3, 9/10, and 12/13. Data were normalized to Actb and represent 3 independent experiments, each performed in triplicate. Note the difference in signal between 2/3 probe and 9/10 probe in GT cells. † P

    Journal: The Journal of Clinical Investigation

    Article Title: LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

    doi: 10.1172/JCI38061

    Figure Lengend Snippet: Confirmation of Lpcat1 GT in ES cells and generation of mice. ( A ) Top: Diagram of Lpcat1 locus. Boxes denote exons, and intervening lines denote introns. The β-geo selection cassette encoding a β-galactosidase/neomycin fusion protein was inserted in intron 9, approximately 400 bp 3′ of exon 9. f1, f2, and r2 denote primers used to confirm the presence of GT cassette in the Lpcat1 locus by qPCR analysis. Bottom: PCR analysis of the Lpcat1 locus in GT ES cells or parental ES cells (129J/Ola) using 2 separate primer sets. The presence of amplicons in GT ES cells with the absence of signal in parental cells demonstrates trapping of the Lpcat1 locus at exon 9. Vertical line indicates discontinuous lanes in the same gel. ( B ) qPCR analysis of GT ES cells. cDNA from GT ES cells (GT) and parental cells (WT) was subjected to qPCR analysis using 3 separate exon-spanning TaqMan primer/probe sets for Lpcat1 as depicted in the bottom panel: 2/3, 9/10, and 12/13. Data were normalized to Actb and represent 3 independent experiments, each performed in triplicate. Note the difference in signal between 2/3 probe and 9/10 probe in GT cells. † P

    Article Snippet: Total RNA for qPCR was isolated and reverse transcribed into cDNA by standard methods. qPCR was performed using TaqMan primer/probe sets (Applied Biosystems) specific for Sftpc (assay ID: Mm00488144_m1), Sftpb (assay ID: Mm00455681_m1), Abca3 (assay ID: Mm00550501_m1), Lpcat1 exons 2/3 (assay ID: Mm00628177_m1), Lpcat1 exons 9/10 (assay ID: Mm00461015_m1), Lpcat1 exons 12/13 (assay ID: Mm01340302_g1), Hspa5 (assay ID: Mm00517691), Fasn (assay ID: Mm01253300_g1), and Scd1 (assay ID: Mm00772290_m1) and Actb (part number: 4352933E) as an internal control.

    Techniques: Mouse Assay, Selection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    ATF4 expression promotes RET ubiquitination. (A and B) HEK 293T cells were transfected with the indicated plasmids, and western blot (WB) analysis was performed with the indicated antibodies. (C) Western blot analysis of TT cells infected with the lentivirus harboring ATF4 with the indicated antibodies. (D) RNA was isolated from cells treated as in (C) and subjected to real-time PCR using ATF4 or RET Taqman primers probes. (E) MTC cells expressing lentiviral-ATF4 were treated with MG132 (4 h), and denatured extracts were immunoprecipitated with an anti-RET antibody followed by western blot analysis with antiubiquitin antibody. (F) ATF4-shRNA (clones of 74 and 76) and nontargeting shRNA (Con) are treated with tunicamycin (2 μg/mL) for 24 h, and western blot analysis was performed with the indicated antibodies. (G) ATF4 expression inhibits RET signaling. MTC cells expressing lentiviral-ATF4 were immunoblotted with the indicated antibodies. IP, immunoprecipitation.

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: ATF4 Targets RET for Degradation and Is a Candidate Tumor Suppressor Gene in Medullary Thyroid Cancer

    doi: 10.1210/jc.2016-2878

    Figure Lengend Snippet: ATF4 expression promotes RET ubiquitination. (A and B) HEK 293T cells were transfected with the indicated plasmids, and western blot (WB) analysis was performed with the indicated antibodies. (C) Western blot analysis of TT cells infected with the lentivirus harboring ATF4 with the indicated antibodies. (D) RNA was isolated from cells treated as in (C) and subjected to real-time PCR using ATF4 or RET Taqman primers probes. (E) MTC cells expressing lentiviral-ATF4 were treated with MG132 (4 h), and denatured extracts were immunoprecipitated with an anti-RET antibody followed by western blot analysis with antiubiquitin antibody. (F) ATF4-shRNA (clones of 74 and 76) and nontargeting shRNA (Con) are treated with tunicamycin (2 μg/mL) for 24 h, and western blot analysis was performed with the indicated antibodies. (G) ATF4 expression inhibits RET signaling. MTC cells expressing lentiviral-ATF4 were immunoblotted with the indicated antibodies. IP, immunoprecipitation.

    Article Snippet: Total RNA and complementary DNA were prepared with use of a Cell-to-CT Kit, and quantitative real-time polymerase chain reaction (PCR) was performed with use of TaqMan primer probes (Thermo Fisher Scientific, Waltham, MA) and hypoxanthine phosphoribosyltransferase as the control.

    Techniques: Expressing, Transfection, Western Blot, Infection, Isolation, Real-time Polymerase Chain Reaction, Immunoprecipitation, shRNA

    Differentially expressed microRNAs in the serum samples Eight differentially expressed microRNAs in the discovery, including miR-7-2-3p A. , miR-4651 B. , miR-127-3p C. , miR-192-5p D. , miR-382-5p E. , miR-10b-5p F. , miR-532-3p G. , and miR-16-5p H. , were further analyzed using TaqMan-PCR method in the training stage. The relative levels of microRNA expression were calculated according to 2 −ΔCT method ( see Materials and Methods ). The microRNA data are shown as box plots, with horizontal lines representing the median, the bottom and the top of the boxes representing the 25th and 75th percentiles, respectively. We compared expression data between groups using the Mann-Whitney U test.

    Journal: Oncotarget

    Article Title: Diagnostic and prognostic potential of serum microRNA-4651 for patients with hepatocellular carcinoma related to aflatoxin B1

    doi: 10.18632/oncotarget.16027

    Figure Lengend Snippet: Differentially expressed microRNAs in the serum samples Eight differentially expressed microRNAs in the discovery, including miR-7-2-3p A. , miR-4651 B. , miR-127-3p C. , miR-192-5p D. , miR-382-5p E. , miR-10b-5p F. , miR-532-3p G. , and miR-16-5p H. , were further analyzed using TaqMan-PCR method in the training stage. The relative levels of microRNA expression were calculated according to 2 −ΔCT method ( see Materials and Methods ). The microRNA data are shown as box plots, with horizontal lines representing the median, the bottom and the top of the boxes representing the 25th and 75th percentiles, respectively. We compared expression data between groups using the Mann-Whitney U test.

    Article Snippet: PCR reactions were run in a 5-μL final volume containing 1 TaqMAN Universal Master Mix II (cat#4440041, Applied Biosystems), 1 × TaqMan microRNA probes and primers (cat#4427975, Applied Biosystems), and about 15 ng of cDNA.

    Techniques: Polymerase Chain Reaction, Expressing, MANN-WHITNEY

    Hypoxia-induced modification of PLK promoter methylation in HCC cells. (a) Promoter methylation status of the plks examined in HCC-derived cells HepG2 and Hep3B; U = unmethylated, M = methylated. Fully methylated HeLa DNA was used as a positive control (+M), no template was added to the negative control (−M). (b) Post hypoxia, PLK4 transcripts were assessed via qPCR in RNA extracted from HCC cells. All qPCR data is representative of the mean value of three independent experiments and error bars represent +/− SD. (c) PLK protein levels were examined post treatment from whole cell lysates. Actin was used as a loading control. (−) represents lysates from untreated cells, (+) lysates from cells grown in the presence of hypoxia. (d) Quantification of protein levels using densitometry. Levels have been normalized to the respective untreated controls. Data is representative of the mean value of three independent experiments and error bars represent +/− SD. (e) The fold change of PLK1 transcripts as determined by qPCR. Values normalized to the respective untreated sample. (f) PLK2 and PLK3 analyzed and fold changed determine by normalization to the respective untreated samples. (g) Hif1α transcripts post hypoxia were determine by real-time PCR using a Taqman probe.

    Journal: PLoS ONE

    Article Title: p53-Dependent and Cell Specific Epigenetic Regulation of the Polo-like kinases under Oxidative Stress

    doi: 10.1371/journal.pone.0087918

    Figure Lengend Snippet: Hypoxia-induced modification of PLK promoter methylation in HCC cells. (a) Promoter methylation status of the plks examined in HCC-derived cells HepG2 and Hep3B; U = unmethylated, M = methylated. Fully methylated HeLa DNA was used as a positive control (+M), no template was added to the negative control (−M). (b) Post hypoxia, PLK4 transcripts were assessed via qPCR in RNA extracted from HCC cells. All qPCR data is representative of the mean value of three independent experiments and error bars represent +/− SD. (c) PLK protein levels were examined post treatment from whole cell lysates. Actin was used as a loading control. (−) represents lysates from untreated cells, (+) lysates from cells grown in the presence of hypoxia. (d) Quantification of protein levels using densitometry. Levels have been normalized to the respective untreated controls. Data is representative of the mean value of three independent experiments and error bars represent +/− SD. (e) The fold change of PLK1 transcripts as determined by qPCR. Values normalized to the respective untreated sample. (f) PLK2 and PLK3 analyzed and fold changed determine by normalization to the respective untreated samples. (g) Hif1α transcripts post hypoxia were determine by real-time PCR using a Taqman probe.

    Article Snippet: Real time PCRs were carried out on an ABI 7300 machine using Taqman gene expression probes for mouse Plk1 , Plk4 , and HIF1α ; and human PLK1-PLK4 , and HIF1α (Applied biosystems).

    Techniques: Modification, Methylation, Derivative Assay, Positive Control, Negative Control, Real-time Polymerase Chain Reaction

    Downregulation of expression levels of miR-17, miR-20a, and miR-106a in promyelocytic cells during SIRPα protein induction by cell differentiation agents. A–C, Induction of SIRPα protein but not SIRPα mRNA by TPA (30 nmol/L) in promyelocytic HL-60 and U937 cells. Note that the SIRPα protein level (Fig 1, A , upper panel , and B ) was significantly increased by TPA, whereas the SIRPα mRNA level (Fig 1, A , lower panel , and C ) was not altered. D, Microarray analysis of changes in miRNA expression in TPA-differentiated HL-60 cells. E, TaqMan probe–based quantitative RT-PCR validation of differentially expressed miRNAs. F and G, Inverse correlation between SIRPα protein levels (Fig 1, F ) and levels of miR-17/20a/106a (Fig 1, G ) in HL-60 cells during RA-induced differentiation process. Data represent means ± SDs of 3 independent experiments performed in triplicate. * P

    Journal: The Journal of allergy and clinical immunology

    Article Title: MicroRNA-17/20a/106a modulate macrophage inflammatory responses through targeting signal-regulatory protein α

    doi: 10.1016/j.jaci.2013.02.005

    Figure Lengend Snippet: Downregulation of expression levels of miR-17, miR-20a, and miR-106a in promyelocytic cells during SIRPα protein induction by cell differentiation agents. A–C, Induction of SIRPα protein but not SIRPα mRNA by TPA (30 nmol/L) in promyelocytic HL-60 and U937 cells. Note that the SIRPα protein level (Fig 1, A , upper panel , and B ) was significantly increased by TPA, whereas the SIRPα mRNA level (Fig 1, A , lower panel , and C ) was not altered. D, Microarray analysis of changes in miRNA expression in TPA-differentiated HL-60 cells. E, TaqMan probe–based quantitative RT-PCR validation of differentially expressed miRNAs. F and G, Inverse correlation between SIRPα protein levels (Fig 1, F ) and levels of miR-17/20a/106a (Fig 1, G ) in HL-60 cells during RA-induced differentiation process. Data represent means ± SDs of 3 independent experiments performed in triplicate. * P

    Article Snippet: Quantitative RT-PCR was performed with TaqMan miRNA probes (Applied Biosystems).

    Techniques: Expressing, Cell Differentiation, Microarray, Quantitative RT-PCR

    Disturbed miRNA expression in osteosarcoma tissue samples. The expression level of eight candidate miRNAs was detected in individual samples using TaqMan miRNA reverse transcription-quantitative polymerase chain reaction. Statistical analyses were performed to analyze the overall trend of each miRNA in all osteosarcoma tissue samples. U6 was used as an internal reference among the different samples and to normalize for experimental error. * P

    Journal: Molecular Medicine Reports

    Article Title: miR-542-3p overexpression is associated with enhanced osteosarcoma cell proliferation and migration ability by targeting Van Gogh-like 2

    doi: 10.3892/mmr.2014.2777

    Figure Lengend Snippet: Disturbed miRNA expression in osteosarcoma tissue samples. The expression level of eight candidate miRNAs was detected in individual samples using TaqMan miRNA reverse transcription-quantitative polymerase chain reaction. Statistical analyses were performed to analyze the overall trend of each miRNA in all osteosarcoma tissue samples. U6 was used as an internal reference among the different samples and to normalize for experimental error. * P

    Article Snippet: TaqMan Universal PCR Master mix with miRNA-specific TaqMan MGB probes (Applied Biosystems, Foster City, CA, USA) was used to amplify the cDNA.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Periostin increases in gingival healing following gingivectomy. ( A ) Gingival tissues dissected from rats were assessed for POSTN gene expression. Isoform analysis using RT-PCR demonstrated the presence of 2 isoforms of POSTN . ( B ) Analysis of the wild-type isoform of POSTN using Taqman PCR showed periostin mRNA peaked at day 7 post wounding. Data represents mean fold gene expressions ± s.d. relative to control day 0 of 3 independent rats. Data was analyzed via one-way ANOVA (* p

    Journal: Scientific Reports

    Article Title: Fibronectin synthesis, but not α-smooth muscle expression, is regulated by periostin in gingival healing through FAK/JNK signaling

    doi: 10.1038/s41598-018-35805-6

    Figure Lengend Snippet: Periostin increases in gingival healing following gingivectomy. ( A ) Gingival tissues dissected from rats were assessed for POSTN gene expression. Isoform analysis using RT-PCR demonstrated the presence of 2 isoforms of POSTN . ( B ) Analysis of the wild-type isoform of POSTN using Taqman PCR showed periostin mRNA peaked at day 7 post wounding. Data represents mean fold gene expressions ± s.d. relative to control day 0 of 3 independent rats. Data was analyzed via one-way ANOVA (* p

    Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed on 50 ng of total RNA using TaqMan qScriptTM One-Step qRT-PCR Kit (Quanta; Gaithersburg, MD, USA) and gene-specific TaqMan probes (Applied Biosystems; Carlsbad, CA, USA) under following conditions: 48 °C for 30 minutes followed by 90 °C for 10 minutes and 40 cycles of 95 °C for 9 seconds and 60 °C for 1 minute using 7900 Real Time PCR system (Applied Biosystems).

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

    High-density SNP mapping in the HBB locus. (a) Distribution of 103 SNPs in the HBB locus interrogated by the Illumina Omni1-Quad chip and TaqMan assay. Globin genes are indicated by boxes. The schematic is not drawn to scale. Abbreviations: LCR, locus

    Journal: Experimental Biology and Medicine

    Article Title: Original Research: A case-control genome-wide association study identifies genetic modifiers of fetal hemoglobin in sickle cell disease

    doi: 10.1177/1535370216642047

    Figure Lengend Snippet: High-density SNP mapping in the HBB locus. (a) Distribution of 103 SNPs in the HBB locus interrogated by the Illumina Omni1-Quad chip and TaqMan assay. Globin genes are indicated by boxes. The schematic is not drawn to scale. Abbreviations: LCR, locus

    Article Snippet: For the HBB locus haplotype analysis, genotype data for SNPs rs2855121 and rs2855122 were confirmed using TaqMan® assay probes (ThermoFisher Scientific, Grand Island, NY) for real-time PCR detection.

    Techniques: Chromatin Immunoprecipitation, TaqMan Assay

    Comparison of multiplex and singleplex assays with TaqMan probe‐based detection. (a) multiplex amplification with mixed TaqMan oligos, mixed TaqMan probes, and mixed target DNA as template. (b–f) Singleplex TaqMan amplification signals when species‐specific TaqMan oligos, species‐specific TaqMan probes, and specific target DNA were used (lines with markers) relative to multiplex amplification signal when mixed TaqMan oligos, mixed TaqMan probes, and mixed target DNA were used (straight lines)

    Journal: Clinical and Experimental Dental Research

    Article Title: Multiplex real‐time PCR detection and relative quantification of periodontal pathogens

    doi: 10.1002/cre2.37

    Figure Lengend Snippet: Comparison of multiplex and singleplex assays with TaqMan probe‐based detection. (a) multiplex amplification with mixed TaqMan oligos, mixed TaqMan probes, and mixed target DNA as template. (b–f) Singleplex TaqMan amplification signals when species‐specific TaqMan oligos, species‐specific TaqMan probes, and specific target DNA were used (lines with markers) relative to multiplex amplification signal when mixed TaqMan oligos, mixed TaqMan probes, and mixed target DNA were used (straight lines)

    Article Snippet: The TaqMan probes were initially optimized in a singleplex assay using Bio‐Rad CFX96 touch thermocycler.

    Techniques: Multiplex Assay, Amplification

    Amplification plots of the GABA-A receptor α 2 -subunit in the TaqMan real-time PCR assay

    Journal: Journal of neuroscience methods

    Article Title: The Optimization of TaqMan Real-Time RT-PCR Assay for Transcriptional Profiling of GABA-A Receptor Subunit Plasticity

    doi: 10.1016/j.jneumeth.2009.04.016

    Figure Lengend Snippet: Amplification plots of the GABA-A receptor α 2 -subunit in the TaqMan real-time PCR assay

    Article Snippet: The PCR primers and TaqMan probe specific for each GABA-A receptor subunit gene and GAPDH were designed using Beacon Designer software (Bio-Rad Inc., Hercules, CA).

    Techniques: Amplification, Real-time Polymerase Chain Reaction

    TTP regulation of CXCR4 mRNA and protein. ( A ) Quantitative PCR (qPCR) quantification of CXCR4 mRNA associated with TTP protein. MDA-MB-231 cells were transfected with TTP or C124R expression plasmids for 24h. Cells were lysed, and TTP and C124R proteins were immunoprecipitated using anti-TTP or normal IgG control antibody. Quantification of associated CXCR4 mRNA was performed by qPCR using a FAM-labeled human CXCR4 Taqman expression probe and normalized to a VIC-labeled GAPDH probe. Data are from one experiment representative of two independent experiments * P

    Journal: Carcinogenesis

    Article Title: Posttranscriptional control of the chemokine receptor CXCR4 expression in cancer cells

    doi: 10.1093/carcin/bgu080

    Figure Lengend Snippet: TTP regulation of CXCR4 mRNA and protein. ( A ) Quantitative PCR (qPCR) quantification of CXCR4 mRNA associated with TTP protein. MDA-MB-231 cells were transfected with TTP or C124R expression plasmids for 24h. Cells were lysed, and TTP and C124R proteins were immunoprecipitated using anti-TTP or normal IgG control antibody. Quantification of associated CXCR4 mRNA was performed by qPCR using a FAM-labeled human CXCR4 Taqman expression probe and normalized to a VIC-labeled GAPDH probe. Data are from one experiment representative of two independent experiments * P

    Article Snippet: Quantitative PCR was performed as multiplex reactions in a C1000 Touch thermal cycler (Bio-Rad, Hercules, CA) using FAM-labeled TaqMan probes (Applied Biosystems, Foster City, CA) for uPA (PLAU), HuR (ELAV1), enhanced green fluorescent protein (EGFP) or human and mouse CXCR4.

    Techniques: Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Transfection, Expressing, Immunoprecipitation, Labeling

    Accumulation of spontaneous tumorigenic Kras mutations in Sirt2 -deficient mice. (a) Accumulation of tumorigenic Kras mutations during inflammation-coupled neoplasm. Mouse pancreas genomic DNA from wild-type and Sirt2 KO, with and without caerulein-treatment, were analyzed for Kras G12D or Kras G12V mutations by competitive allele-specific TaqMan PCR. The table shows the summary of analysis for percentage of mice positive for mutations (46 wild-type mice, 56 Sirt2 KO mice) after pancreatitis. (b) Detail description of wild-type and Sirt2 KO mice with Kras G12D and Kras G12V mutations. (c) Bar graph representation of ( b ). ( d ) Immunostaining detection of KRAS-G12D mutant protein in mouse pancreas. Mice pancreas tissues were fixed, embedded in paraffin, and immuno-histochemical staining with anti- KRAS-G12D antibody was performed. Representative images are shown. Bars indicate 100 µm.

    Journal: Scientific Reports

    Article Title: Loss of Sirt2 increases and prolongs a caerulein-induced pancreatitis permissive phenotype and induces spontaneous oncogenic Kras mutations in mice

    doi: 10.1038/s41598-018-34792-y

    Figure Lengend Snippet: Accumulation of spontaneous tumorigenic Kras mutations in Sirt2 -deficient mice. (a) Accumulation of tumorigenic Kras mutations during inflammation-coupled neoplasm. Mouse pancreas genomic DNA from wild-type and Sirt2 KO, with and without caerulein-treatment, were analyzed for Kras G12D or Kras G12V mutations by competitive allele-specific TaqMan PCR. The table shows the summary of analysis for percentage of mice positive for mutations (46 wild-type mice, 56 Sirt2 KO mice) after pancreatitis. (b) Detail description of wild-type and Sirt2 KO mice with Kras G12D and Kras G12V mutations. (c) Bar graph representation of ( b ). ( d ) Immunostaining detection of KRAS-G12D mutant protein in mouse pancreas. Mice pancreas tissues were fixed, embedded in paraffin, and immuno-histochemical staining with anti- KRAS-G12D antibody was performed. Representative images are shown. Bars indicate 100 µm.

    Article Snippet: Genomic DNA isolated from mouse pancreas were subjected to TaqMan PCR with custom TaqMan probes specific for mouse Kras mutation G12D or G12V (Applied Biosystems).

    Techniques: Mouse Assay, Polymerase Chain Reaction, Immunostaining, Mutagenesis, Staining

    Schematic of competition assays used to measure relative fitness levels between two strains. ( a ) Overview of life history of the standard reference N2 strain since its isolation from the wild. Derived alleles in npr-1 and glb-5 arose and fixed after 1957 and before 1969 when methods for cryopreservation were developed. These two alleles were identified for their role in changing foraging behavior on bacterial lawns from social to solitary behavior. ( b ) Schematic of pairwise competition experiments used throughout the paper to quantify fitness differences between two strains. ( c ) Relative proportion of each strain as ascertained by Droplet Digital PCR using a custom TaqMan probe (dots) is used to estimate the relative fitness between the two strains (line). ( d ) Silent mutations were edited into the 90 th or 92 nd amino acid of the dpy-10 gene using CRISPR/Cas9 to create a common SNV for Droplet Digital PCR. We refer to these as barcoded strains. ( e ) Competition experiments between the parent strain (top) and the same strain containing one of the silent mutations. We display the result from each competition experiment as a single dot overlaid on top of a boxplot showing the mean, first, and third quartiles of all replicates.

    Journal: eLife

    Article Title: Changes to social feeding behaviors are not sufficient for fitness gains of the Caenorhabditis elegans N2 reference strain

    doi: 10.7554/eLife.38675

    Figure Lengend Snippet: Schematic of competition assays used to measure relative fitness levels between two strains. ( a ) Overview of life history of the standard reference N2 strain since its isolation from the wild. Derived alleles in npr-1 and glb-5 arose and fixed after 1957 and before 1969 when methods for cryopreservation were developed. These two alleles were identified for their role in changing foraging behavior on bacterial lawns from social to solitary behavior. ( b ) Schematic of pairwise competition experiments used throughout the paper to quantify fitness differences between two strains. ( c ) Relative proportion of each strain as ascertained by Droplet Digital PCR using a custom TaqMan probe (dots) is used to estimate the relative fitness between the two strains (line). ( d ) Silent mutations were edited into the 90 th or 92 nd amino acid of the dpy-10 gene using CRISPR/Cas9 to create a common SNV for Droplet Digital PCR. We refer to these as barcoded strains. ( e ) Competition experiments between the parent strain (top) and the same strain containing one of the silent mutations. We display the result from each competition experiment as a single dot overlaid on top of a boxplot showing the mean, first, and third quartiles of all replicates.

    Article Snippet: To quantify the relative proportion of each strain, we used a digital PCR based approach using a custom TaqMan probe (Applied Biosciences).

    Techniques: Isolation, Derivative Assay, Digital PCR, CRISPR

    (a) Schematic representation of the agr operon organisation and regulation in Staphylococcus aureus . (b) Schematic representation showing single-site chromosomal integration of GFP transcriptional fusion reporters for P2 and P3. (c) Sequence of P2 and P3 promoter regions used to generate the gfp transcriptional fusions for P2 and P3 (the consensus −10 and −35 sites are outlined, the transcription start sites are shown with arrows and the RBS is underlined. (d) Sheep blood agar haemolysis assay with S. aureus USA300 WT (1), USA300 agrA ::Tn [ agrA transposon mutant from NARSA library (Fey et al ., 2013 )] (2), USA300 agr IR P3-GFP (3) and USA300 agr IR P2-GFP (4). (e) Graphs showing relative hld , agrA and gyrB mRNA levels in 16 h cultures of S. aureus USA300 WT and USA300 agr IR P3-GFP as determined by Taqman qRT-PCR. Values are shown for each gene with respect to USA300 WT levels. (f) Graph showing growth curves (OD 600 ) of S. aureus USA300 WT , USA300 agr IR P2-GFP and USA300 agr IR P3-GFP strains grown in TSB. (g) Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] and GFP-FU as a function of growth (OD 600 ) over time for S. aureus USA300 agr IR P2-GFP and USA300 agr IR P3-GFP strains grown in TSB. (h) As in (g) but with S. aureus SH1000 agr IR P2-GFP , SH1000 agr IR P3-GFP , SH1001 agr IR P2-GFP and SH1001 agr IR P3-GFP strains grown in TSB. Data for (d–h) were obtained from three biological replicates

    Journal: Fems Microbiology Letters

    Article Title: Transcriptional downregulation of agr expression in Staphylococcus aureus during growth in human serum can be overcome by constitutively active mutant forms of the sensor kinase AgrC

    doi: 10.1111/1574-6968.12309

    Figure Lengend Snippet: (a) Schematic representation of the agr operon organisation and regulation in Staphylococcus aureus . (b) Schematic representation showing single-site chromosomal integration of GFP transcriptional fusion reporters for P2 and P3. (c) Sequence of P2 and P3 promoter regions used to generate the gfp transcriptional fusions for P2 and P3 (the consensus −10 and −35 sites are outlined, the transcription start sites are shown with arrows and the RBS is underlined. (d) Sheep blood agar haemolysis assay with S. aureus USA300 WT (1), USA300 agrA ::Tn [ agrA transposon mutant from NARSA library (Fey et al ., 2013 )] (2), USA300 agr IR P3-GFP (3) and USA300 agr IR P2-GFP (4). (e) Graphs showing relative hld , agrA and gyrB mRNA levels in 16 h cultures of S. aureus USA300 WT and USA300 agr IR P3-GFP as determined by Taqman qRT-PCR. Values are shown for each gene with respect to USA300 WT levels. (f) Graph showing growth curves (OD 600 ) of S. aureus USA300 WT , USA300 agr IR P2-GFP and USA300 agr IR P3-GFP strains grown in TSB. (g) Graphs showing GFP expression [as GFP fluorescence units (GFP-FU)] and GFP-FU as a function of growth (OD 600 ) over time for S. aureus USA300 agr IR P2-GFP and USA300 agr IR P3-GFP strains grown in TSB. (h) As in (g) but with S. aureus SH1000 agr IR P2-GFP , SH1000 agr IR P3-GFP , SH1001 agr IR P2-GFP and SH1001 agr IR P3-GFP strains grown in TSB. Data for (d–h) were obtained from three biological replicates

    Article Snippet: Real-time quantitative reverse transcription PCR (qRT-PCR) Details of RNA extraction and cDNA synthesis can be found in the Data S1. qRT-PCR was performed using primers and Taqman probes corresponding to hld (delta toxin), agrA and gyrB (gyrase B) with QPCR core kit, no ROX (Eurogentec) according to the manufacturer's instructions.

    Techniques: Sequencing, Haemolysis Assay, Mutagenesis, Quantitative RT-PCR, Expressing, Fluorescence