taqman fast universal pcr master mix  (Thermo Fisher)


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    TaqMan Fast Universal PCR Master Mix 2X
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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 Fast Universal PCR Master Mix and added additional capabilities for your gene expression analysis TaqMan Fast Universal Master Mix 2x No AmpErase UNG delivers results in 40 minutes for 40 cycles of PCR in a 20 µL reaction volume using the Applied Biosystems 7900HT and Fast 7500 Fast Real Time PCR Systems The optimized formulation contains AmpliTaq Fast DNA Polymerase UP a highly purified DNA polymerase designed to allow instant hot start minimizing non specific product formation and allowing room temperature reaction setup Additionally a proprietary ROX dye serves as a passive internal reference to normalize non PCR related fluorescence fluctuations for superb precision on Applied Biosystems real time PCR instruments • Convenience Everything you need for TaqMan based qPCR amplification and detection in a single tube format TaqMan Fast Universal PCR Master Mix contains all of the components excluding the template and primers for superior real time qPCR • Compatibility TaqMan Fast Universal PCR Master Mix 2x No AmpErase UNG is compatible with TaqMan Gene Expression assays
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    4352042
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    Enzymes & Master Mixes for Real-Time PCR|Fast Real-Time PCR|PCR & Real-Time PCR|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Gene Expression Analysis & Genotyping
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    Structured Review

    Thermo Fisher taqman fast universal pcr master mix
    Effects of SPIB knock-down on resistant cells. a Conformation of partial silencing of SPIB on gene level. The relative mRNA expression of SPIB was assessed in the cells using <t>TaqMan</t> probe based <t>RT-PCR</t> and revealed a knock of gene expression with 50% compared to un-knocked control cells. The data are normalised to the S18 reference gene. b Protein levels of SPIB measured with western blot 48 h after transfection with siRNA. GAPDH was used as loading control. Representative figure of three biological replicates. c Normalised proliferation for Z138 cells grown in cytarabine containing medium, measured by incorporation of [methyl-14C]-thymidine 72 h after knock of SPIB using siRNA. Each data point represents a mean value of triplicates and error bars show SD. * = p
    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 Fast Universal PCR Master Mix and added additional capabilities for your gene expression analysis TaqMan Fast Universal Master Mix 2x No AmpErase UNG delivers results in 40 minutes for 40 cycles of PCR in a 20 µL reaction volume using the Applied Biosystems 7900HT and Fast 7500 Fast Real Time PCR Systems The optimized formulation contains AmpliTaq Fast DNA Polymerase UP a highly purified DNA polymerase designed to allow instant hot start minimizing non specific product formation and allowing room temperature reaction setup Additionally a proprietary ROX dye serves as a passive internal reference to normalize non PCR related fluorescence fluctuations for superb precision on Applied Biosystems real time PCR instruments • Convenience Everything you need for TaqMan based qPCR amplification and detection in a single tube format TaqMan Fast Universal PCR Master Mix contains all of the components excluding the template and primers for superior real time qPCR • Compatibility TaqMan Fast Universal PCR Master Mix 2x No AmpErase UNG is compatible with TaqMan Gene Expression assays
    https://www.bioz.com/result/taqman fast universal pcr master mix/product/Thermo Fisher
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    taqman fast universal pcr master mix - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity"

    Article Title: Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4346-1

    Effects of SPIB knock-down on resistant cells. a Conformation of partial silencing of SPIB on gene level. The relative mRNA expression of SPIB was assessed in the cells using TaqMan probe based RT-PCR and revealed a knock of gene expression with 50% compared to un-knocked control cells. The data are normalised to the S18 reference gene. b Protein levels of SPIB measured with western blot 48 h after transfection with siRNA. GAPDH was used as loading control. Representative figure of three biological replicates. c Normalised proliferation for Z138 cells grown in cytarabine containing medium, measured by incorporation of [methyl-14C]-thymidine 72 h after knock of SPIB using siRNA. Each data point represents a mean value of triplicates and error bars show SD. * = p
    Figure Legend Snippet: Effects of SPIB knock-down on resistant cells. a Conformation of partial silencing of SPIB on gene level. The relative mRNA expression of SPIB was assessed in the cells using TaqMan probe based RT-PCR and revealed a knock of gene expression with 50% compared to un-knocked control cells. The data are normalised to the S18 reference gene. b Protein levels of SPIB measured with western blot 48 h after transfection with siRNA. GAPDH was used as loading control. Representative figure of three biological replicates. c Normalised proliferation for Z138 cells grown in cytarabine containing medium, measured by incorporation of [methyl-14C]-thymidine 72 h after knock of SPIB using siRNA. Each data point represents a mean value of triplicates and error bars show SD. * = p

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

    Verification of dCK mRNA expression in gene microarray samples. The relative mRNA expression of dCK was assessed in the different Z138 subclones using TaqMan probe based RT-PCR and revealed down-regulation in both Z138-CytES and Z138-CytR cells compared to Z138-CytNS cells. The data are normalised to the S18 reference gene, and the reference sample, replicate A of the Z138-CytNS for each run. Bars represent relative quantity ± SEM of three technical replicates. * = p
    Figure Legend Snippet: Verification of dCK mRNA expression in gene microarray samples. The relative mRNA expression of dCK was assessed in the different Z138 subclones using TaqMan probe based RT-PCR and revealed down-regulation in both Z138-CytES and Z138-CytR cells compared to Z138-CytNS cells. The data are normalised to the S18 reference gene, and the reference sample, replicate A of the Z138-CytNS for each run. Bars represent relative quantity ± SEM of three technical replicates. * = p

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

    2) Product Images from "Defining the Transcriptional and Cellular Landscape of Type 1 Diabetes in the NOD Mouse"

    Article Title: Defining the Transcriptional and Cellular Landscape of Type 1 Diabetes in the NOD Mouse

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059701

    Quantitative RT-PCR validation of microarray data. ( A ) Quantitative RT-PCR was performed using SYBR green detection for the indicated genes. Bars show the mean (log 2 ) +/− S.E.M. of at least three independent experimental replicates from 3–6 biological replicates per group. All data is represented relative to the expression of actin (ΔC t ). In order to facilitate visualization on a log 2 scale, values were transformed as indicated on the y-axis label. ( B ) Microarray results for the same genes interrogated in ( A ). ( C ) Taqman qPCR quantification of pan-IFNα, Ifnb1 , or Ifng throughout diabetogenesis. Bars represent the mean of the normalized probe intensity +/− S.E.M. of 3–6 biological replicates per group. Asterisks indicate statistical significance (P
    Figure Legend Snippet: Quantitative RT-PCR validation of microarray data. ( A ) Quantitative RT-PCR was performed using SYBR green detection for the indicated genes. Bars show the mean (log 2 ) +/− S.E.M. of at least three independent experimental replicates from 3–6 biological replicates per group. All data is represented relative to the expression of actin (ΔC t ). In order to facilitate visualization on a log 2 scale, values were transformed as indicated on the y-axis label. ( B ) Microarray results for the same genes interrogated in ( A ). ( C ) Taqman qPCR quantification of pan-IFNα, Ifnb1 , or Ifng throughout diabetogenesis. Bars represent the mean of the normalized probe intensity +/− S.E.M. of 3–6 biological replicates per group. Asterisks indicate statistical significance (P

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

    3) Product Images from "Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA"

    Article Title: Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.05.014

    Editing of CEP290 Transcripts Occurs in HEK293T after Transfection with PTMs (A) Illustration of adeno-associated virus genome arrangement of a CEP290 5′ PTM with binding domain 07 (PTM_07) with either no poly-adenylation signal (NPA) or with a bovine growth hormone poly-adenylation signal (PA). ITR, inverted terminal repeat; CMV, cytomegalovirus promoter; PCDS, partial coding DNA sequence of CEP290 ; 5′ SS, 5′ splice site. (B) qPCR was performed on HEK293T transfected with a plasmid encoding GFP as a transfection control or the plasmids in (A). TaqMan probes were designed to the junctions indicated: CEP290 exons 26 and 27 (hEx26-hEx27), a region within the 5′ codon optimized partial coding DNA sequence (coPCDS), or the novel junction of 5′ coPCDS and endogenous Homo sapiens exon 27 to signify trans -splicing (coPCDS-hEx27). Significant variation within replicates for coPCDS-hEx27 was present because the Ct was crossed at 35 cycles with either PTM; however, no amplification was observed through 40 cycles in GFP-treated samples. Samples were standardized to β-2-microglobin and normalized to NPA. Error bars are relative quantity minimum and maximum 95% confidence intervals. (C) Agarose gel electrophoresis of one of the replicate reactions from (B). (D) Densitometry analysis of the bands in (C). (E) Sanger sequencing following TOPO-cloning of the PCR product comprising the coPCDS-hEx27 junction visualized in (C). Nucleotide differences between Homo sapiens and codon-optimized CEP290 are noted by asterisks. The junction between the 5′ coPCDS and endogenous exon 27 is marked by a vertical dashed line.
    Figure Legend Snippet: Editing of CEP290 Transcripts Occurs in HEK293T after Transfection with PTMs (A) Illustration of adeno-associated virus genome arrangement of a CEP290 5′ PTM with binding domain 07 (PTM_07) with either no poly-adenylation signal (NPA) or with a bovine growth hormone poly-adenylation signal (PA). ITR, inverted terminal repeat; CMV, cytomegalovirus promoter; PCDS, partial coding DNA sequence of CEP290 ; 5′ SS, 5′ splice site. (B) qPCR was performed on HEK293T transfected with a plasmid encoding GFP as a transfection control or the plasmids in (A). TaqMan probes were designed to the junctions indicated: CEP290 exons 26 and 27 (hEx26-hEx27), a region within the 5′ codon optimized partial coding DNA sequence (coPCDS), or the novel junction of 5′ coPCDS and endogenous Homo sapiens exon 27 to signify trans -splicing (coPCDS-hEx27). Significant variation within replicates for coPCDS-hEx27 was present because the Ct was crossed at 35 cycles with either PTM; however, no amplification was observed through 40 cycles in GFP-treated samples. Samples were standardized to β-2-microglobin and normalized to NPA. Error bars are relative quantity minimum and maximum 95% confidence intervals. (C) Agarose gel electrophoresis of one of the replicate reactions from (B). (D) Densitometry analysis of the bands in (C). (E) Sanger sequencing following TOPO-cloning of the PCR product comprising the coPCDS-hEx27 junction visualized in (C). Nucleotide differences between Homo sapiens and codon-optimized CEP290 are noted by asterisks. The junction between the 5′ coPCDS and endogenous exon 27 is marked by a vertical dashed line.

    Techniques Used: Transfection, Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Clone Assay, Polymerase Chain Reaction

    Editing of Mini- CEP290 Transcripts Occurs In Vivo following Sub-retinal Injection of 7m8AAV-5′ PTMs (A) Diagram of the CEP290 intron 26 mini-gene. The mini-gene is driven by the murine Rhodopsin promoter (mRho). CEP290 exons 25 and 26 are joined and followed by the complete intron 26-27 and exon 27. An A > G mutation corresponding to c.2991+1655 was included to assess exon X splicing in the model. An amino-terminal Myc and a carboxy-terminal FLAG tag were added to flank the mini-gene. Additionally, an internal ribosomal entry site (IRES) was added to drive expression of EGFP and is terminated by a bovine growth hormone poly-adenylation signal sequence (pA). (B) Immunofluorescence staining of a retinal cross-section from a mini- CEP290 mouse. OS, outer segment. ONL, outer nuclear layer. INL, inner nuclear layer. Scale bar is 50 μm. (C) Illustration of potential splicing outcomes within the mini- CEP290 mouse. Canonical cis- splicing would result in a predicted 24.3-kDa peptide. Alternative cis- splicing with exon X would yield a predicted non-sense media decay transcript encoding a truncated peptide with Myc at 17.1 kDa and no FLAG translation. Trans -splicing with a 5′ PTM would replace the amino-terminal Myc and generate a 124.1-kDa peptide with a FLAG tag. Arrow pairs indicate locations of PCR primers used to detect specific splicing events. (D) Representative western blot images of Myc and FLAG at 24.3 kDa showing OD and OS lanes per animal for each contralateral treatment cohort. (E) Densitometry quantification showing the mean log 10 values of each contralateral treatment cohort. Samples were standardized to α-tubulin. Error bars are SEM. Sample sizes as indicated. Individual animal data is available in Figure S1 A. (F) qPCR from cDNA generated by RNA extracts from whole eyes of mini- CEP290 mice. TaqMan probes were designed to the junctions of Homo sapiens exon 27 and FLAG to detect total expression of the mini-gene (left) to a region within the PCDS to detect total expression of the PTM (center) or to the novel junction of codon-optimized CEP290 PCDS and Homo sapiens CEP290 exon 27 (right). Values from treatment-matched samples were averaged as biological groups, standardized to murine β-2-microglobin and normalized to PTM_NBD. p
    Figure Legend Snippet: Editing of Mini- CEP290 Transcripts Occurs In Vivo following Sub-retinal Injection of 7m8AAV-5′ PTMs (A) Diagram of the CEP290 intron 26 mini-gene. The mini-gene is driven by the murine Rhodopsin promoter (mRho). CEP290 exons 25 and 26 are joined and followed by the complete intron 26-27 and exon 27. An A > G mutation corresponding to c.2991+1655 was included to assess exon X splicing in the model. An amino-terminal Myc and a carboxy-terminal FLAG tag were added to flank the mini-gene. Additionally, an internal ribosomal entry site (IRES) was added to drive expression of EGFP and is terminated by a bovine growth hormone poly-adenylation signal sequence (pA). (B) Immunofluorescence staining of a retinal cross-section from a mini- CEP290 mouse. OS, outer segment. ONL, outer nuclear layer. INL, inner nuclear layer. Scale bar is 50 μm. (C) Illustration of potential splicing outcomes within the mini- CEP290 mouse. Canonical cis- splicing would result in a predicted 24.3-kDa peptide. Alternative cis- splicing with exon X would yield a predicted non-sense media decay transcript encoding a truncated peptide with Myc at 17.1 kDa and no FLAG translation. Trans -splicing with a 5′ PTM would replace the amino-terminal Myc and generate a 124.1-kDa peptide with a FLAG tag. Arrow pairs indicate locations of PCR primers used to detect specific splicing events. (D) Representative western blot images of Myc and FLAG at 24.3 kDa showing OD and OS lanes per animal for each contralateral treatment cohort. (E) Densitometry quantification showing the mean log 10 values of each contralateral treatment cohort. Samples were standardized to α-tubulin. Error bars are SEM. Sample sizes as indicated. Individual animal data is available in Figure S1 A. (F) qPCR from cDNA generated by RNA extracts from whole eyes of mini- CEP290 mice. TaqMan probes were designed to the junctions of Homo sapiens exon 27 and FLAG to detect total expression of the mini-gene (left) to a region within the PCDS to detect total expression of the PTM (center) or to the novel junction of codon-optimized CEP290 PCDS and Homo sapiens CEP290 exon 27 (right). Values from treatment-matched samples were averaged as biological groups, standardized to murine β-2-microglobin and normalized to PTM_NBD. p

    Techniques Used: In Vivo, Injection, Mutagenesis, FLAG-tag, Expressing, Sequencing, Immunofluorescence, Staining, Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Generated, Mouse Assay

    4) Product Images from "Cytomegalovirus pp71 Protein Is Expressed in Human Glioblastoma and Promotes Pro-Angiogenic Signaling by Activation of Stem Cell Factor"

    Article Title: Cytomegalovirus pp71 Protein Is Expressed in Human Glioblastoma and Promotes Pro-Angiogenic Signaling by Activation of Stem Cell Factor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068176

    Detection of pp71 RNA and protein in primary glioma specimens. A: RNA was extracted from 20 different primary brain tissues, synthesized into cDNA, and amplified using pp71 and Rab14-specific PCR primers. RNA from U251 glioma cells mock infected or infected with HCMV Towne strain and commercially available RNA samples from normal fetal and adult human brain were used as controls. The N.C. negative control PCR contained water in place of cDNA. B : Several cDNA samples described in A were analyzed by TaqMan using primers and probes specific for the pp71 gene and normalized to Rab14. Copy number was determined using Ad169 viral DNA standard curve. C : Western blot analysis for pp71 from 10 different primary brain tissues. Lysates from normal human astrocytes and mock-infected or HCMV-infected U87 glioma cells were used as controls. D : Cells from 3 freshly resected GBM were sorted using CD133 labeled antibody and analyzed by RT-PCR for pp71 and Rab14. Negative control (NC) is PCR performed with water instead of cDNA. E : HCMV-infected primary GBM cell line (3832) was sorted using CD133 labeled antibody at 72 hours post-infection and analyzed by TaqMan for pp71 compared to a mock-treated control. The relative expression of pp71 normalized to Rab14 is displayed for 3 independent experiments, *p
    Figure Legend Snippet: Detection of pp71 RNA and protein in primary glioma specimens. A: RNA was extracted from 20 different primary brain tissues, synthesized into cDNA, and amplified using pp71 and Rab14-specific PCR primers. RNA from U251 glioma cells mock infected or infected with HCMV Towne strain and commercially available RNA samples from normal fetal and adult human brain were used as controls. The N.C. negative control PCR contained water in place of cDNA. B : Several cDNA samples described in A were analyzed by TaqMan using primers and probes specific for the pp71 gene and normalized to Rab14. Copy number was determined using Ad169 viral DNA standard curve. C : Western blot analysis for pp71 from 10 different primary brain tissues. Lysates from normal human astrocytes and mock-infected or HCMV-infected U87 glioma cells were used as controls. D : Cells from 3 freshly resected GBM were sorted using CD133 labeled antibody and analyzed by RT-PCR for pp71 and Rab14. Negative control (NC) is PCR performed with water instead of cDNA. E : HCMV-infected primary GBM cell line (3832) was sorted using CD133 labeled antibody at 72 hours post-infection and analyzed by TaqMan for pp71 compared to a mock-treated control. The relative expression of pp71 normalized to Rab14 is displayed for 3 independent experiments, *p

    Techniques Used: Synthesized, Amplification, Polymerase Chain Reaction, Infection, Negative Control, Western Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing

    5) Product Images from "PPARγ Expression Is Diminished in Macrophages of Recurrent Miscarriage Placentas"

    Article Title: PPARγ Expression Is Diminished in Macrophages of Recurrent Miscarriage Placentas

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19071872

    Results of PPARγ mRNA expression analysis with TaqMan RT-PCR from trophoblastic tissue. PPARγ mRNA expression was significantly downregulated in the miscarriage groups (SM, 15 cases, p = 0.01) and RM, 16 cases, p = 0.004)) compared to the healthy controls (15 cases). This bar graph shows the mean of relative PPARγ expression; therefore, the presentation of error bars is not appropriate.
    Figure Legend Snippet: Results of PPARγ mRNA expression analysis with TaqMan RT-PCR from trophoblastic tissue. PPARγ mRNA expression was significantly downregulated in the miscarriage groups (SM, 15 cases, p = 0.01) and RM, 16 cases, p = 0.004)) compared to the healthy controls (15 cases). This bar graph shows the mean of relative PPARγ expression; therefore, the presentation of error bars is not appropriate.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    6) Product Images from "Bone marrow adipocytes promote tumor growth in bone via FABP4-dependent mechanisms"

    Article Title: Bone marrow adipocytes promote tumor growth in bone via FABP4-dependent mechanisms

    Journal: Oncotarget

    doi:

    Chronic exposure to low dose Adipo CM increases invasiveness and induces FABP4, HMOX-1 and IL-1β expression A: Schematic of long-term culture conditions. Cells are exposed to gradually increasing concentration of Adipo CM (gradient of 5-25%) and maintained in 25% Adipo CM over multiple passages. B: DIC images showing invasive morphology of PC3 cells after chronic Adipo CM exposure. C: Taqman RT PCR for FABP4, IL-1β, and HMOX-1 expression of PC3 cells. Data are normalized to 18S and shown as increase relative to control cultures. D: Invasion assay of PC3 cells treated long-term with control or Adipo CM media for 12 passages. Prior to assay cells were serum starved, seeded in serum free media on top of rBM-coated filter and allowed to invade towards DMEM with 10% FBS for 48 hours. Top panels: Diff-Quik stained invasion filters. Bottom panels: Quantification results showing numbers of invaded cells.
    Figure Legend Snippet: Chronic exposure to low dose Adipo CM increases invasiveness and induces FABP4, HMOX-1 and IL-1β expression A: Schematic of long-term culture conditions. Cells are exposed to gradually increasing concentration of Adipo CM (gradient of 5-25%) and maintained in 25% Adipo CM over multiple passages. B: DIC images showing invasive morphology of PC3 cells after chronic Adipo CM exposure. C: Taqman RT PCR for FABP4, IL-1β, and HMOX-1 expression of PC3 cells. Data are normalized to 18S and shown as increase relative to control cultures. D: Invasion assay of PC3 cells treated long-term with control or Adipo CM media for 12 passages. Prior to assay cells were serum starved, seeded in serum free media on top of rBM-coated filter and allowed to invade towards DMEM with 10% FBS for 48 hours. Top panels: Diff-Quik stained invasion filters. Bottom panels: Quantification results showing numbers of invaded cells.

    Techniques Used: Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Invasion Assay, Diff-Quik, Staining

    PPARγ is involved in Adipo CM-induced expression of FABP4, IL-1β, and HMOX-1 A: Western blot analysis of intracellular FABP4, IL-1β and HMOX-1 expression in PC3 cells grown in the absence or presence of rosiglitazone (ROSI, 1 and 10μM), Adipo CM, and Adipo CM in the presence of PPARγ antagonist GW9662 (1 and 10μM). B: Densitometric analysis of FABP4 (top panel), IL-1β (middle panel) and HMOX-1 (bottom panel) bands normalized to β-actin. C: Taqman RT PCR analysis of FABP4, IL-1β and HMOX-1 expression in PC3 cells grown in the absence or presence of rosiglitazone (1 and 10μM), Adipo CM, and Adipo CM in the presence of PPARγ antagonist GW9662 (1 and 10μM). Similar increase in FABP4, IL-1β and HMOX-1 expression is observed with ROSI and Adipo CM and expression of FABP4 and IL-1β is reduced with GW9662. D: PPARγ gene expression (Taqman RT PCR) in PC3 cells exposed to Adipo CM. Data are normalized to 18S and GUSB and show a decrease in PPARγ levels with Adipo CM; E: DNA binding assay in nuclear fractions from control and Adipo CM-treated PC3 cells showing reduced PPARγ DNA binding upon Adipo CM treatment. Adipo CM-suppressed gene expression (F) and DNA-binding activity (G) of PPARγ can be restored by GW9662 antagonist or FABP4 inhibitor; Values * p
    Figure Legend Snippet: PPARγ is involved in Adipo CM-induced expression of FABP4, IL-1β, and HMOX-1 A: Western blot analysis of intracellular FABP4, IL-1β and HMOX-1 expression in PC3 cells grown in the absence or presence of rosiglitazone (ROSI, 1 and 10μM), Adipo CM, and Adipo CM in the presence of PPARγ antagonist GW9662 (1 and 10μM). B: Densitometric analysis of FABP4 (top panel), IL-1β (middle panel) and HMOX-1 (bottom panel) bands normalized to β-actin. C: Taqman RT PCR analysis of FABP4, IL-1β and HMOX-1 expression in PC3 cells grown in the absence or presence of rosiglitazone (1 and 10μM), Adipo CM, and Adipo CM in the presence of PPARγ antagonist GW9662 (1 and 10μM). Similar increase in FABP4, IL-1β and HMOX-1 expression is observed with ROSI and Adipo CM and expression of FABP4 and IL-1β is reduced with GW9662. D: PPARγ gene expression (Taqman RT PCR) in PC3 cells exposed to Adipo CM. Data are normalized to 18S and GUSB and show a decrease in PPARγ levels with Adipo CM; E: DNA binding assay in nuclear fractions from control and Adipo CM-treated PC3 cells showing reduced PPARγ DNA binding upon Adipo CM treatment. Adipo CM-suppressed gene expression (F) and DNA-binding activity (G) of PPARγ can be restored by GW9662 antagonist or FABP4 inhibitor; Values * p

    Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, DNA Binding Assay, Binding Assay, Activity Assay

    Bone marrow adipocyte-supplied lipids stimulate proliferation and invasion of prostate tumor cells and upregulate genes involved in fatty acid transport A: DNA assay results for cells grown in collagen I gels in the absence or presence of Adipo CM. B: Images of invasion filters coated with reconstituted basement membrane; cells in the absence (Control) or presence of media conditioned by Bone Marrow Mesenchymal cells (BMMC CM) or adipocytes (Adipo CM) were allowed to invade toward DMEM containing 10% FBS for 48 hours C: Quantitation results of invaded cells shown as % control ± SD. D: BODIPY 493/503 staining of lipid droplets (green) for control and Adipo CM-treated PC3 cells. E: Quantitation of lipid fluorescence (Metamorph). F: Taqman RT-PCR analysis (Life Technologies) of lipid droplet-associated genes: CD36, FABP4 and Perilipin 2 expression in PC3 cells +/− Adipo CM. Data are normalized to 18S. G: BODIPY 493/503 staining of lipid droplets (green) in adipocytes cultured alone (left panels) or in transwell with PC3 cells (right panels). H: Taqman RT-PCR analysis (Life Technologies) of adipocyte-specific gene (FABP4, Adiponectin and Resistin expression) in bone marrow adipocytes cultured alone or in transwell with PC3 cells. Data are normalized to HPRT1.
    Figure Legend Snippet: Bone marrow adipocyte-supplied lipids stimulate proliferation and invasion of prostate tumor cells and upregulate genes involved in fatty acid transport A: DNA assay results for cells grown in collagen I gels in the absence or presence of Adipo CM. B: Images of invasion filters coated with reconstituted basement membrane; cells in the absence (Control) or presence of media conditioned by Bone Marrow Mesenchymal cells (BMMC CM) or adipocytes (Adipo CM) were allowed to invade toward DMEM containing 10% FBS for 48 hours C: Quantitation results of invaded cells shown as % control ± SD. D: BODIPY 493/503 staining of lipid droplets (green) for control and Adipo CM-treated PC3 cells. E: Quantitation of lipid fluorescence (Metamorph). F: Taqman RT-PCR analysis (Life Technologies) of lipid droplet-associated genes: CD36, FABP4 and Perilipin 2 expression in PC3 cells +/− Adipo CM. Data are normalized to 18S. G: BODIPY 493/503 staining of lipid droplets (green) in adipocytes cultured alone (left panels) or in transwell with PC3 cells (right panels). H: Taqman RT-PCR analysis (Life Technologies) of adipocyte-specific gene (FABP4, Adiponectin and Resistin expression) in bone marrow adipocytes cultured alone or in transwell with PC3 cells. Data are normalized to HPRT1.

    Techniques Used: Quantitation Assay, Staining, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture

    FABP4, IL-1β, and HMOX-1 are expressed in tumor cells exposed to adipocyte-derived factors in vitro and in bone tumors in vivo A: Genes upregulated in PC3 cells exposed to Adipo CM in vitro as detected by Taqman RT PCR Human Inflammation Array (Life Technologies). Data are normalized to HPRT1 and GUSB and shown as fold increase relative to control. B, C: Taqman RT-PCR of FABP4, IL-1β, and HMOX-1 in PC3 bone tumors (B) and PC3 subcutaneous tumors (C) from LFD and HFD mice. Data are normalized to HPRT1 and GUSB and shown as fold increase relative to LFD.
    Figure Legend Snippet: FABP4, IL-1β, and HMOX-1 are expressed in tumor cells exposed to adipocyte-derived factors in vitro and in bone tumors in vivo A: Genes upregulated in PC3 cells exposed to Adipo CM in vitro as detected by Taqman RT PCR Human Inflammation Array (Life Technologies). Data are normalized to HPRT1 and GUSB and shown as fold increase relative to control. B, C: Taqman RT-PCR of FABP4, IL-1β, and HMOX-1 in PC3 bone tumors (B) and PC3 subcutaneous tumors (C) from LFD and HFD mice. Data are normalized to HPRT1 and GUSB and shown as fold increase relative to LFD.

    Techniques Used: Derivative Assay, In Vitro, In Vivo, Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    7) Product Images from "Exonic duplication CNV of NDRG1 associated with autosomal-recessive HMSN-Lom/CMT4D"

    Article Title: Exonic duplication CNV of NDRG1 associated with autosomal-recessive HMSN-Lom/CMT4D

    Journal: Genetics in medicine : official journal of the American College of Medical Genetics

    doi: 10.1038/gim.2013.155

    Sequence analysis for cDNA and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) study for NDRG1 ( a ) Sanger sequence results of cDNA from the control and affected individuals. In healthy individuals, exon 8 is followed by exon 9 and in affected individuals exon 8 is followed by exon 6. ( b ) Analysis of the resultant sequence at the amino acid level. The duplication causes a frame shift mutation, altering the amino acid sequence for several residues before a premature termination codon. ( c ) Expression levels in blood were measured by quantitative RT-PCR using the TaqMan gene expression assay in triplicate and normalized to TBP . We observed a decrease in expression, 0.12- to 0.18-fold in patient samples (BAB3724 and BAB4149) and 0.6- to 0.61-fold in carriers (BAB4146 and BAB4147), compared with normal expression controls. We found an obviously decreased expression in both patients and carriers compared with control healthy individuals. * P
    Figure Legend Snippet: Sequence analysis for cDNA and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) study for NDRG1 ( a ) Sanger sequence results of cDNA from the control and affected individuals. In healthy individuals, exon 8 is followed by exon 9 and in affected individuals exon 8 is followed by exon 6. ( b ) Analysis of the resultant sequence at the amino acid level. The duplication causes a frame shift mutation, altering the amino acid sequence for several residues before a premature termination codon. ( c ) Expression levels in blood were measured by quantitative RT-PCR using the TaqMan gene expression assay in triplicate and normalized to TBP . We observed a decrease in expression, 0.12- to 0.18-fold in patient samples (BAB3724 and BAB4149) and 0.6- to 0.61-fold in carriers (BAB4146 and BAB4147), compared with normal expression controls. We found an obviously decreased expression in both patients and carriers compared with control healthy individuals. * P

    Techniques Used: Sequencing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Expressing, Quantitative RT-PCR

    8) Product Images from "Increases in transient receptor potential vanilloid-1 mRNA and protein in primary afferent neurons stimulated by protein kinase C and their possible role in neurogenic inflammation"

    Article Title: Increases in transient receptor potential vanilloid-1 mRNA and protein in primary afferent neurons stimulated by protein kinase C and their possible role in neurogenic inflammation

    Journal: Journal of neuroscience research

    doi: 10.1002/jnr.21844

    CAP-evoked expressions of TRPV 1 and CGRP mRNA ( A and B ) and the effects of a TPRV 1 antagonist and a PKC inhibitor ( C and D ). Relative quantification (RQ) of TRPV 1 and CGRP mRNA level was done by TaqMan® Fast Universal PCR Master Mix and calculated
    Figure Legend Snippet: CAP-evoked expressions of TRPV 1 and CGRP mRNA ( A and B ) and the effects of a TPRV 1 antagonist and a PKC inhibitor ( C and D ). Relative quantification (RQ) of TRPV 1 and CGRP mRNA level was done by TaqMan® Fast Universal PCR Master Mix and calculated

    Techniques Used: Polymerase Chain Reaction

    9) Product Images from "Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response"

    Article Title: Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response

    Journal: Journal of Virology

    doi: 10.1128/JVI.03055-15

    HFt cells stably transduced with DOX-inducible eYFP.PIAS4 wild-type or mutant protein expression constructs. (A) The illustration depicts the conserved PIAS4 domains (gray boxes) mutated within eYFP.PIAS4 (eYFP.P4) ( Table 1 ). (B) Confocal images show the nuclear expression of eYFP, eYFP.P4 wild-type (wt) or mutant proteins (as indicated) in transgenic HFt cells after 8 h of induction (+DOX) or not (−DOX). Nuclei were visualized by DAPI (blue). (C) Western blot shows the levels of eYFP.PIAS4 wild-type (eYFP.P4 wt) or endogenous PIAS4 (PIAS4) in transgenic HFt cells after different lengths of DOX induction, as indicated. Membranes were probed for PIAS4 or actin as a loading control. The asterisk indicates a PIAS4 polyclonal antibody cross-reacting band. (D) A bar graph shows the average level of eYFP.P4 wt relative to endogenous PIAS4 in transgenic cells after various lengths of DOX induction. The intensities of PIAS4 protein bands from Western blots as in panel C were quantitated and normalized to their respective loading controls. Normalized levels of eYFP.P4 wt are expressed relative to the normalized levels of endogenous PIAS4 at each time. Means and standard deviations (SD) are shown ( n = 3). (E) Whole-cell lysates collected from transgenic HFt cells 8 h post-DOX induction were analyzed by Western blotting for the levels of eYFP and eYFP.PIAS4 wild-type or mutant protein (as indicated) expression. Membranes were probed for eYFP or actin as a loading control. (F) Western blot shows the levels of eYFP.PIAS4 wild-type or catalytically inactive mutant (C342A) proteins at different times after translational inhibition. Expression of eYFP.P4 wt or eYFP.P4.C342A was DOX induced for 4 h prior to the replacement of DOX with the translation inhibitor cycloheximide (CHX) in the presence (+) or absence (−) of MG132. Whole-cell lysates were harvested at 0, 2, 4, 6, or 8 h following CHX addition. Membranes were probed with eYFP or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (G) A bar graph shows the average relative levels of eYFP.PIAS4 wild-type or catalytically inactive mutant proteins following translation inhibition. The intensities of protein bands corresponding to eYFP.P4 quantitated from Western blots as in panel F were normalized to their respective loading controls and expressed relative to the normalized levels at 0 h of CHX treatment (1.0). Means and SEM are shown ( n = 3). (H) A bar graph shows the average relative levels of PIAS4 mRNA in transgenic cells that express eYFP.P4 wild-type or catalytically inactive mutant proteins. Total RNA was isolated after 4, 8, or 24 h of DOX induction. The levels of PIAS4 mRNA were determined using the TaqMan system of quantitative RT-PCR. Values were normalized to GAPDH expression using the threshold cycle (ΔΔ C T ) method and are expressed relative to the normalized level of PIAS4 mRNA at 0 h of DOX induction ( 1 ). Values represent the averages from two independent experiments. (I) A bar graph shows the average relative level of endogenous PIAS4 following translation inhibition. HFt cells were treated with CHX in the presence (+) or absence (−) of MG132, and whole-cell lysates were collected at 2, 4, or 8 h after CHX addition. Intensities of the protein bands corresponding to PIAS4 were quantitated and normalized to their respective loading controls and are expressed relative to the normalized level of PIAS4 at 0 h of CHX treatment (1.0). Means and SEM are shown ( n ≥ 3).
    Figure Legend Snippet: HFt cells stably transduced with DOX-inducible eYFP.PIAS4 wild-type or mutant protein expression constructs. (A) The illustration depicts the conserved PIAS4 domains (gray boxes) mutated within eYFP.PIAS4 (eYFP.P4) ( Table 1 ). (B) Confocal images show the nuclear expression of eYFP, eYFP.P4 wild-type (wt) or mutant proteins (as indicated) in transgenic HFt cells after 8 h of induction (+DOX) or not (−DOX). Nuclei were visualized by DAPI (blue). (C) Western blot shows the levels of eYFP.PIAS4 wild-type (eYFP.P4 wt) or endogenous PIAS4 (PIAS4) in transgenic HFt cells after different lengths of DOX induction, as indicated. Membranes were probed for PIAS4 or actin as a loading control. The asterisk indicates a PIAS4 polyclonal antibody cross-reacting band. (D) A bar graph shows the average level of eYFP.P4 wt relative to endogenous PIAS4 in transgenic cells after various lengths of DOX induction. The intensities of PIAS4 protein bands from Western blots as in panel C were quantitated and normalized to their respective loading controls. Normalized levels of eYFP.P4 wt are expressed relative to the normalized levels of endogenous PIAS4 at each time. Means and standard deviations (SD) are shown ( n = 3). (E) Whole-cell lysates collected from transgenic HFt cells 8 h post-DOX induction were analyzed by Western blotting for the levels of eYFP and eYFP.PIAS4 wild-type or mutant protein (as indicated) expression. Membranes were probed for eYFP or actin as a loading control. (F) Western blot shows the levels of eYFP.PIAS4 wild-type or catalytically inactive mutant (C342A) proteins at different times after translational inhibition. Expression of eYFP.P4 wt or eYFP.P4.C342A was DOX induced for 4 h prior to the replacement of DOX with the translation inhibitor cycloheximide (CHX) in the presence (+) or absence (−) of MG132. Whole-cell lysates were harvested at 0, 2, 4, 6, or 8 h following CHX addition. Membranes were probed with eYFP or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (G) A bar graph shows the average relative levels of eYFP.PIAS4 wild-type or catalytically inactive mutant proteins following translation inhibition. The intensities of protein bands corresponding to eYFP.P4 quantitated from Western blots as in panel F were normalized to their respective loading controls and expressed relative to the normalized levels at 0 h of CHX treatment (1.0). Means and SEM are shown ( n = 3). (H) A bar graph shows the average relative levels of PIAS4 mRNA in transgenic cells that express eYFP.P4 wild-type or catalytically inactive mutant proteins. Total RNA was isolated after 4, 8, or 24 h of DOX induction. The levels of PIAS4 mRNA were determined using the TaqMan system of quantitative RT-PCR. Values were normalized to GAPDH expression using the threshold cycle (ΔΔ C T ) method and are expressed relative to the normalized level of PIAS4 mRNA at 0 h of DOX induction ( 1 ). Values represent the averages from two independent experiments. (I) A bar graph shows the average relative level of endogenous PIAS4 following translation inhibition. HFt cells were treated with CHX in the presence (+) or absence (−) of MG132, and whole-cell lysates were collected at 2, 4, or 8 h after CHX addition. Intensities of the protein bands corresponding to PIAS4 were quantitated and normalized to their respective loading controls and are expressed relative to the normalized level of PIAS4 at 0 h of CHX treatment (1.0). Means and SEM are shown ( n ≥ 3).

    Techniques Used: Stable Transfection, Transduction, Mutagenesis, Expressing, Construct, Transgenic Assay, Western Blot, Inhibition, Isolation, Quantitative RT-PCR

    Endogenous PIAS4 contributes to the cellular restriction of ICP0-null mutant HSV-1. (A) Western blots show the levels of PIAS4 or PML in transgenic HFt cells that express short hairpin RNAs against PIAS4 (shPIAS4), PML (shPML), or a control sequence (shCtrl). Membranes were probed for PIAS4, PML, or actin as a loading control. (B) A bar graph shows the average relative levels of PIAS4 or PML mRNA in transgenic HFt cells that express shCtrl, shPIAS4, or shPML. PIAS4 or PML mRNA levels were determined using the TaqMan system of quantitative RT-PCR. Values normalized to 18S expression using the ΔΔ C T method are expressed relative to cells that expressed shCtrl (1.0). Values represent means and SD from three independent rounds of RT using mRNA isolated from one representative experiment. (C) A bar graph shows the average relative plaque formation efficiency (PFE) of wild-type or ICP0-null mutant (ΔICP0) HSV-1 in transgenic cells that express shCtrl, shPIAS4, or shPML, as indicated. The PFE for each strain in cells that express shPIAS4 or shPML was normalized to the respective PFE in cells that express shCtrl ( 1 ). Means and SD are shown ( n ≥ 3). (D and E) Western blots show the levels of viral protein expression during wild-type or ICP0-null mutant (ΔICP0) HSV-1 infection of transgenic HFt cells that express shCtrl or shPIAS4. Transgenic cells mock infected or infected with 10 PFU of wild-type (D) or ICP0-null mutant (E) HSV-1 per cell in the presence (+) or absence (−) of MG132 were harvested at 3, 6, or 9 hpi. Membranes were probed for ICP4, ICP0, UL42, or VP5 to monitor the progression of infection or actin as a loading control. Molecular mass markers are shown, in kilodaltons. (F) A bar graph shows the average relative levels of PML or PIAS4 mRNA in transgenic cells that express shCtrl, shPIAS4, or shPML alone or in combination, as indicated. Analysis was performed as for panel B. Neo, neomycin resistance; Puro, puromycin resistance. (G) A bar graph shows the average relative PFE of wild-type or ICP0-null mutant HSV-1 in transgenic HFt cells that express shCtrl, shPIAS4, or shPML alone or in combination, as indicated. PFE analysis was done as described for panel C. Means and SD are shown ( n ≥ 3).
    Figure Legend Snippet: Endogenous PIAS4 contributes to the cellular restriction of ICP0-null mutant HSV-1. (A) Western blots show the levels of PIAS4 or PML in transgenic HFt cells that express short hairpin RNAs against PIAS4 (shPIAS4), PML (shPML), or a control sequence (shCtrl). Membranes were probed for PIAS4, PML, or actin as a loading control. (B) A bar graph shows the average relative levels of PIAS4 or PML mRNA in transgenic HFt cells that express shCtrl, shPIAS4, or shPML. PIAS4 or PML mRNA levels were determined using the TaqMan system of quantitative RT-PCR. Values normalized to 18S expression using the ΔΔ C T method are expressed relative to cells that expressed shCtrl (1.0). Values represent means and SD from three independent rounds of RT using mRNA isolated from one representative experiment. (C) A bar graph shows the average relative plaque formation efficiency (PFE) of wild-type or ICP0-null mutant (ΔICP0) HSV-1 in transgenic cells that express shCtrl, shPIAS4, or shPML, as indicated. The PFE for each strain in cells that express shPIAS4 or shPML was normalized to the respective PFE in cells that express shCtrl ( 1 ). Means and SD are shown ( n ≥ 3). (D and E) Western blots show the levels of viral protein expression during wild-type or ICP0-null mutant (ΔICP0) HSV-1 infection of transgenic HFt cells that express shCtrl or shPIAS4. Transgenic cells mock infected or infected with 10 PFU of wild-type (D) or ICP0-null mutant (E) HSV-1 per cell in the presence (+) or absence (−) of MG132 were harvested at 3, 6, or 9 hpi. Membranes were probed for ICP4, ICP0, UL42, or VP5 to monitor the progression of infection or actin as a loading control. Molecular mass markers are shown, in kilodaltons. (F) A bar graph shows the average relative levels of PML or PIAS4 mRNA in transgenic cells that express shCtrl, shPIAS4, or shPML alone or in combination, as indicated. Analysis was performed as for panel B. Neo, neomycin resistance; Puro, puromycin resistance. (G) A bar graph shows the average relative PFE of wild-type or ICP0-null mutant HSV-1 in transgenic HFt cells that express shCtrl, shPIAS4, or shPML alone or in combination, as indicated. PFE analysis was done as described for panel C. Means and SD are shown ( n ≥ 3).

    Techniques Used: Mutagenesis, Western Blot, Transgenic Assay, Sequencing, Quantitative RT-PCR, Expressing, Isolation, Infection

    10) Product Images from "Differential Roles of Peroxisome Proliferator-Activated Receptor-α and Receptor-γ on Renal Crystal Formation in Hyperoxaluric Rodents"

    Article Title: Differential Roles of Peroxisome Proliferator-Activated Receptor-α and Receptor-γ on Renal Crystal Formation in Hyperoxaluric Rodents

    Journal: PPAR Research

    doi: 10.1155/2016/9605890

    Proinflammatory gene expression in model rat kidneys. (a) Distribution of proinflammatory-related gene expression in rat kidneys obtained on day 14. OPN, osteopontin; MCP-1, monocyte chemoattractant protein-1. Magnification: 400x. Scale bar = 50 μ m. (b) Gene expression was assessed by qRT-PCR using TaqMan assays ( n = 6 for all groups). Data are presented as mean ± SE. ∗ p
    Figure Legend Snippet: Proinflammatory gene expression in model rat kidneys. (a) Distribution of proinflammatory-related gene expression in rat kidneys obtained on day 14. OPN, osteopontin; MCP-1, monocyte chemoattractant protein-1. Magnification: 400x. Scale bar = 50 μ m. (b) Gene expression was assessed by qRT-PCR using TaqMan assays ( n = 6 for all groups). Data are presented as mean ± SE. ∗ p

    Techniques Used: Expressing, Quantitative RT-PCR

    Anti-inflammatory and oxidative stress gene expression in model rat kidneys. (a) Distribution of anti-inflammatory and oxidative stress-related gene expressions in rat kidneys obtained on day 14. APN, adiponectin; SOD1, superoxide dismutase 1. Magnification: 200x. Scale bar = 100 μ m. (b) Gene expression was measured by qRT-PCR using TaqMan assays ( n = 6 for all groups). Data are presented as the mean ± SE. ∗ p
    Figure Legend Snippet: Anti-inflammatory and oxidative stress gene expression in model rat kidneys. (a) Distribution of anti-inflammatory and oxidative stress-related gene expressions in rat kidneys obtained on day 14. APN, adiponectin; SOD1, superoxide dismutase 1. Magnification: 200x. Scale bar = 100 μ m. (b) Gene expression was measured by qRT-PCR using TaqMan assays ( n = 6 for all groups). Data are presented as the mean ± SE. ∗ p

    Techniques Used: Expressing, Quantitative RT-PCR

    PPAR α and PPAR γ gene expression in model rat kidneys. (a) Gene expression was assessed by qRT-PCR using TaqMan assays ( n = 6 for all groups). Data are presented as mean ± SE. † p
    Figure Legend Snippet: PPAR α and PPAR γ gene expression in model rat kidneys. (a) Gene expression was assessed by qRT-PCR using TaqMan assays ( n = 6 for all groups). Data are presented as mean ± SE. † p

    Techniques Used: Expressing, Quantitative RT-PCR

    Renal CaOx crystal formation and PPAR expression in hyperoxaluric mice. (a) Quantitative estimation of renal CaOx crystals. Crystallization in each kidney section on designed time points was quantified by calculating the ratio (%) of the area containing crystals to that of the entire kidney section by using Image Pro Plus. (b) Evaluation of PPAR α and PPAR γ expression by qRT-PCR using TaqMan assays ( n = 6 for each time point). Control values represent the average on day 0 and data are presented as mean ± SE. ∗ p
    Figure Legend Snippet: Renal CaOx crystal formation and PPAR expression in hyperoxaluric mice. (a) Quantitative estimation of renal CaOx crystals. Crystallization in each kidney section on designed time points was quantified by calculating the ratio (%) of the area containing crystals to that of the entire kidney section by using Image Pro Plus. (b) Evaluation of PPAR α and PPAR γ expression by qRT-PCR using TaqMan assays ( n = 6 for each time point). Control values represent the average on day 0 and data are presented as mean ± SE. ∗ p

    Techniques Used: Expressing, Mouse Assay, Crystallization Assay, Quantitative RT-PCR

    11) Product Images from "Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer"

    Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25535

    Comparison of miRNA expression levels obtained by NGS Illumina sequencing vs. TaqMan PCR. The results of the correlation are visualized as bar diagrams on the left and scatter diagrams with regression lines on the right comparing next generation sequencing (blue/y-axis) and TaqMan fold changes (red/x-axis) for hsa-miR-378a ( A ), hsa-miR-375 ( B ), hsa-miR-21-5p ( C ) and hsa-miR-215-5p ( D ). There was a good correlation of the NGS with the TaqMan results with a strong relationship for hsa-miR-378a (Pearson’s r = 0.9), hsa-miR-375 ( r = 0.77) and hsa-miR-215-5p ( r = 0.75), but only a moderate relationship for hsa-miR-21-5p ( r = 0.53).
    Figure Legend Snippet: Comparison of miRNA expression levels obtained by NGS Illumina sequencing vs. TaqMan PCR. The results of the correlation are visualized as bar diagrams on the left and scatter diagrams with regression lines on the right comparing next generation sequencing (blue/y-axis) and TaqMan fold changes (red/x-axis) for hsa-miR-378a ( A ), hsa-miR-375 ( B ), hsa-miR-21-5p ( C ) and hsa-miR-215-5p ( D ). There was a good correlation of the NGS with the TaqMan results with a strong relationship for hsa-miR-378a (Pearson’s r = 0.9), hsa-miR-375 ( r = 0.77) and hsa-miR-215-5p ( r = 0.75), but only a moderate relationship for hsa-miR-21-5p ( r = 0.53).

    Techniques Used: Expressing, Next-Generation Sequencing, Sequencing, Polymerase Chain Reaction

    12) Product Images from "Human Cytomegalovirus Gene Expression in Long-Term Infected Glioma Stem Cells"

    Article Title: Human Cytomegalovirus Gene Expression in Long-Term Infected Glioma Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116178

    HCMV gene expression is detectable at 15 weeks p.i only in 387 GSC cells. A. 387 GSC cells infected with AD169 virus (MOI = 2) were analyzed for viral gene expression with SYBR Green RT-PCR at 72 hours, 3 weeks and every 2 weeks thereafter up to 15 weeks. B. Taqman validation of a subset of viral genes of AD169-infected 387 cells at 11 weeks p.i. Relative expression levels were obtained by normalizing viral gene expression with expression of cellular gene Rab14. Each sample was run in triplicate, bars represent S.D. C. Comparison of viral gene expression of AD169-infected glioma stem-like cells and cell lines at 72 hours and 5 weeks p.i. 3832 GSC cells were infected with AD169 virus (MOI = 2) and analyzed for viral gene expression with SYBR Green RT-PCR at 72 hours and 5 weeks p.i. These results are displayed alongside 72 hr and 5 week p.i. data for AD169-infected 387, U87, and T98G from Figures 1D–E and Figure 2A .
    Figure Legend Snippet: HCMV gene expression is detectable at 15 weeks p.i only in 387 GSC cells. A. 387 GSC cells infected with AD169 virus (MOI = 2) were analyzed for viral gene expression with SYBR Green RT-PCR at 72 hours, 3 weeks and every 2 weeks thereafter up to 15 weeks. B. Taqman validation of a subset of viral genes of AD169-infected 387 cells at 11 weeks p.i. Relative expression levels were obtained by normalizing viral gene expression with expression of cellular gene Rab14. Each sample was run in triplicate, bars represent S.D. C. Comparison of viral gene expression of AD169-infected glioma stem-like cells and cell lines at 72 hours and 5 weeks p.i. 3832 GSC cells were infected with AD169 virus (MOI = 2) and analyzed for viral gene expression with SYBR Green RT-PCR at 72 hours and 5 weeks p.i. These results are displayed alongside 72 hr and 5 week p.i. data for AD169-infected 387, U87, and T98G from Figures 1D–E and Figure 2A .

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

    13) Product Images from "RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes"

    Article Title: RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00775

    Design and execution of the small-hairpin (sh)RNA screens for murine nuclear cofactors of IL-1 signaling. For screen I, 4–5 shRNAs directed at up to four different nuclear targets per 48-well plate were transfected in duplicates as shown. In screen II, 4–5 shRNAs per nuclear target were pooled and transfected into a single well resulting in screening of 20 nuclear targets per plate (not shown). In both screens, empty vector (pLKO.1) with no insert or an insert encoding a scrambled shRNA sequence (pLKO.1-scr.) were used as controls on each of the plates. Cells transfected with pLKO.1 encoding a GFP cDNA were used to monitor transfection efficiency on each individual plate by fluorescence microscopy (left image) and by phase contrast plus fluorescence microscopy (right image) as shown by the insets. The scale bar is 100 µm. For each screen, 3.5 × 10 4 cells were seeded per well. One day later, 270 ng of DNA were transfected using Lipofectamine LTX plus reagent ® . Cells were selected for 48 h in 1 µg/ml puromycin. Then, half of the cells on each plate were left untreated. The other half was stimulated for 3 h (screen I) or 1 h (screen II) with IL-1α (10 ng/ml). Thereafter, cells were lysed, and RT reactions and preamplifications of cDNAs were performed using the PreAmp Cells-to-Ct TM Kit and gene specific primers for the IL-1-inducible target gene Cxcl2 and the two “housekeeping” genes ActB (screen I only) and Ube2l3 . Finally, preamplified PCR products were subjected to quantitative (q)PCR using Taqman assays and mRNA expressions levels were quantified based on their cycle threshold (ct) value using an ABI7500 instrument. Ct values were used for all further calculations.
    Figure Legend Snippet: Design and execution of the small-hairpin (sh)RNA screens for murine nuclear cofactors of IL-1 signaling. For screen I, 4–5 shRNAs directed at up to four different nuclear targets per 48-well plate were transfected in duplicates as shown. In screen II, 4–5 shRNAs per nuclear target were pooled and transfected into a single well resulting in screening of 20 nuclear targets per plate (not shown). In both screens, empty vector (pLKO.1) with no insert or an insert encoding a scrambled shRNA sequence (pLKO.1-scr.) were used as controls on each of the plates. Cells transfected with pLKO.1 encoding a GFP cDNA were used to monitor transfection efficiency on each individual plate by fluorescence microscopy (left image) and by phase contrast plus fluorescence microscopy (right image) as shown by the insets. The scale bar is 100 µm. For each screen, 3.5 × 10 4 cells were seeded per well. One day later, 270 ng of DNA were transfected using Lipofectamine LTX plus reagent ® . Cells were selected for 48 h in 1 µg/ml puromycin. Then, half of the cells on each plate were left untreated. The other half was stimulated for 3 h (screen I) or 1 h (screen II) with IL-1α (10 ng/ml). Thereafter, cells were lysed, and RT reactions and preamplifications of cDNAs were performed using the PreAmp Cells-to-Ct TM Kit and gene specific primers for the IL-1-inducible target gene Cxcl2 and the two “housekeeping” genes ActB (screen I only) and Ube2l3 . Finally, preamplified PCR products were subjected to quantitative (q)PCR using Taqman assays and mRNA expressions levels were quantified based on their cycle threshold (ct) value using an ABI7500 instrument. Ct values were used for all further calculations.

    Techniques Used: Transfection, Plasmid Preparation, shRNA, Sequencing, Fluorescence, Microscopy, Polymerase Chain Reaction

    14) Product Images from "Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer"

    Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25535

    Comparison of miRNA expression levels obtained by NGS Illumina sequencing vs. TaqMan PCR. The results of the correlation are visualized as bar diagrams on the left and scatter diagrams with regression lines on the right comparing next generation sequencing (blue/y-axis) and TaqMan fold changes (red/x-axis) for hsa-miR-378a ( A ), hsa-miR-375 ( B ), hsa-miR-21-5p ( C ) and hsa-miR-215-5p ( D ). There was a good correlation of the NGS with the TaqMan results with a strong relationship for hsa-miR-378a (Pearson’s r = 0.9), hsa-miR-375 ( r = 0.77) and hsa-miR-215-5p ( r = 0.75), but only a moderate relationship for hsa-miR-21-5p ( r = 0.53).
    Figure Legend Snippet: Comparison of miRNA expression levels obtained by NGS Illumina sequencing vs. TaqMan PCR. The results of the correlation are visualized as bar diagrams on the left and scatter diagrams with regression lines on the right comparing next generation sequencing (blue/y-axis) and TaqMan fold changes (red/x-axis) for hsa-miR-378a ( A ), hsa-miR-375 ( B ), hsa-miR-21-5p ( C ) and hsa-miR-215-5p ( D ). There was a good correlation of the NGS with the TaqMan results with a strong relationship for hsa-miR-378a (Pearson’s r = 0.9), hsa-miR-375 ( r = 0.77) and hsa-miR-215-5p ( r = 0.75), but only a moderate relationship for hsa-miR-21-5p ( r = 0.53).

    Techniques Used: Expressing, Next-Generation Sequencing, Sequencing, Polymerase Chain Reaction

    15) Product Images from "Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA"

    Article Title: Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.05.014

    Editing of CEP290 Transcripts Occurs in HEK293T after Transfection with PTMs (A) Illustration of adeno-associated virus genome arrangement of a CEP290 5′ PTM with binding domain 07 (PTM_07) with either no poly-adenylation signal (NPA) or with a bovine growth hormone poly-adenylation signal (PA). ITR, inverted terminal repeat; CMV, cytomegalovirus promoter; PCDS, partial coding DNA sequence of CEP290 ; 5′ SS, 5′ splice site. (B) qPCR was performed on HEK293T transfected with a plasmid encoding GFP as a transfection control or the plasmids in (A). TaqMan probes were designed to the junctions indicated: CEP290 exons 26 and 27 (hEx26-hEx27), a region within the 5′ codon optimized partial coding DNA sequence (coPCDS), or the novel junction of 5′ coPCDS and endogenous Homo sapiens exon 27 to signify trans -splicing (coPCDS-hEx27). Significant variation within replicates for coPCDS-hEx27 was present because the Ct was crossed at 35 cycles with either PTM; however, no amplification was observed through 40 cycles in GFP-treated samples. Samples were standardized to β-2-microglobin and normalized to NPA. Error bars are relative quantity minimum and maximum 95% confidence intervals. (C) Agarose gel electrophoresis of one of the replicate reactions from (B). (D) Densitometry analysis of the bands in (C). (E) Sanger sequencing following TOPO-cloning of the PCR product comprising the coPCDS-hEx27 junction visualized in (C). Nucleotide differences between Homo sapiens and codon-optimized CEP290 are noted by asterisks. The junction between the 5′ coPCDS and endogenous exon 27 is marked by a vertical dashed line.
    Figure Legend Snippet: Editing of CEP290 Transcripts Occurs in HEK293T after Transfection with PTMs (A) Illustration of adeno-associated virus genome arrangement of a CEP290 5′ PTM with binding domain 07 (PTM_07) with either no poly-adenylation signal (NPA) or with a bovine growth hormone poly-adenylation signal (PA). ITR, inverted terminal repeat; CMV, cytomegalovirus promoter; PCDS, partial coding DNA sequence of CEP290 ; 5′ SS, 5′ splice site. (B) qPCR was performed on HEK293T transfected with a plasmid encoding GFP as a transfection control or the plasmids in (A). TaqMan probes were designed to the junctions indicated: CEP290 exons 26 and 27 (hEx26-hEx27), a region within the 5′ codon optimized partial coding DNA sequence (coPCDS), or the novel junction of 5′ coPCDS and endogenous Homo sapiens exon 27 to signify trans -splicing (coPCDS-hEx27). Significant variation within replicates for coPCDS-hEx27 was present because the Ct was crossed at 35 cycles with either PTM; however, no amplification was observed through 40 cycles in GFP-treated samples. Samples were standardized to β-2-microglobin and normalized to NPA. Error bars are relative quantity minimum and maximum 95% confidence intervals. (C) Agarose gel electrophoresis of one of the replicate reactions from (B). (D) Densitometry analysis of the bands in (C). (E) Sanger sequencing following TOPO-cloning of the PCR product comprising the coPCDS-hEx27 junction visualized in (C). Nucleotide differences between Homo sapiens and codon-optimized CEP290 are noted by asterisks. The junction between the 5′ coPCDS and endogenous exon 27 is marked by a vertical dashed line.

    Techniques Used: Transfection, Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Clone Assay, Polymerase Chain Reaction

    Editing of Mini- CEP290 Transcripts Occurs In Vivo following Sub-retinal Injection of 7m8AAV-5′ PTMs (A) Diagram of the CEP290 intron 26 mini-gene. The mini-gene is driven by the murine Rhodopsin promoter (mRho). CEP290 exons 25 and 26 are joined and followed by the complete intron 26-27 and exon 27. An A > G mutation corresponding to c.2991+1655 was included to assess exon X splicing in the model. An amino-terminal Myc and a carboxy-terminal FLAG tag were added to flank the mini-gene. Additionally, an internal ribosomal entry site (IRES) was added to drive expression of EGFP and is terminated by a bovine growth hormone poly-adenylation signal sequence (pA). (B) Immunofluorescence staining of a retinal cross-section from a mini- CEP290 mouse. OS, outer segment. ONL, outer nuclear layer. INL, inner nuclear layer. Scale bar is 50 μm. (C) Illustration of potential splicing outcomes within the mini- CEP290 mouse. Canonical cis- splicing would result in a predicted 24.3-kDa peptide. Alternative cis- splicing with exon X would yield a predicted non-sense media decay transcript encoding a truncated peptide with Myc at 17.1 kDa and no FLAG translation. Trans -splicing with a 5′ PTM would replace the amino-terminal Myc and generate a 124.1-kDa peptide with a FLAG tag. Arrow pairs indicate locations of PCR primers used to detect specific splicing events. (D) Representative western blot images of Myc and FLAG at 24.3 kDa showing OD and OS lanes per animal for each contralateral treatment cohort. (E) Densitometry quantification showing the mean log 10 A. (F) qPCR from cDNA generated by RNA extracts from whole eyes of mini- CEP290 mice. TaqMan probes were designed to the junctions of Homo sapiens exon 27 and FLAG to detect total expression of the mini-gene (left) to a region within the PCDS to detect total expression of the PTM (center) or to the novel junction of codon-optimized CEP290 PCDS and Homo sapiens CEP290 B.
    Figure Legend Snippet: Editing of Mini- CEP290 Transcripts Occurs In Vivo following Sub-retinal Injection of 7m8AAV-5′ PTMs (A) Diagram of the CEP290 intron 26 mini-gene. The mini-gene is driven by the murine Rhodopsin promoter (mRho). CEP290 exons 25 and 26 are joined and followed by the complete intron 26-27 and exon 27. An A > G mutation corresponding to c.2991+1655 was included to assess exon X splicing in the model. An amino-terminal Myc and a carboxy-terminal FLAG tag were added to flank the mini-gene. Additionally, an internal ribosomal entry site (IRES) was added to drive expression of EGFP and is terminated by a bovine growth hormone poly-adenylation signal sequence (pA). (B) Immunofluorescence staining of a retinal cross-section from a mini- CEP290 mouse. OS, outer segment. ONL, outer nuclear layer. INL, inner nuclear layer. Scale bar is 50 μm. (C) Illustration of potential splicing outcomes within the mini- CEP290 mouse. Canonical cis- splicing would result in a predicted 24.3-kDa peptide. Alternative cis- splicing with exon X would yield a predicted non-sense media decay transcript encoding a truncated peptide with Myc at 17.1 kDa and no FLAG translation. Trans -splicing with a 5′ PTM would replace the amino-terminal Myc and generate a 124.1-kDa peptide with a FLAG tag. Arrow pairs indicate locations of PCR primers used to detect specific splicing events. (D) Representative western blot images of Myc and FLAG at 24.3 kDa showing OD and OS lanes per animal for each contralateral treatment cohort. (E) Densitometry quantification showing the mean log 10 A. (F) qPCR from cDNA generated by RNA extracts from whole eyes of mini- CEP290 mice. TaqMan probes were designed to the junctions of Homo sapiens exon 27 and FLAG to detect total expression of the mini-gene (left) to a region within the PCDS to detect total expression of the PTM (center) or to the novel junction of codon-optimized CEP290 PCDS and Homo sapiens CEP290 B.

    Techniques Used: In Vivo, Injection, Mutagenesis, FLAG-tag, Expressing, Sequencing, Immunofluorescence, Staining, Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Generated, Mouse Assay

    16) Product Images from "Uncoupling Antisense-Mediated Silencing and DNA Methylation in the Imprinted Gnas Cluster"

    Article Title: Uncoupling Antisense-Mediated Silencing and DNA Methylation in the Imprinted Gnas Cluster

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001347

    Nesp is fully expressed from the paternal allele in +/ Nespas-T ex1 . (A) The Nespas-T ex1 allele was generated by targeting and is identical to Nesp-T int2 except that the polyadenylation cassette was inserted in the opposite orientation (Ap) to truncate Nespas. The selection genes were deleted from the Ap- neo targeted allele upon germline transmission by Cre-recombinase mediated excision to generate Nespas-T ex1 . (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (Ap- neo ) cells. The Ap- neo targeted clones were identified by the presence of an 8.5 kb Nde I fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb Avr II fragment with the 5′ external probe. (C) RT-PCR assay in newborn brain showing Nespas is truncated. A primer pair, 3′ of the insertion, detected Nespas whereas a primer pair 5′ of the insertion did not detect Nespas . Symbols + and −, refer to reactions carried out in the presence and absence of reverse transcriptase, respectively. As the Nespas PCR products were weak, it was necessary to blot and probe the Nespas RT-PCR products with appropriate genomic probes. Hprt was included as an amplification control. (D) Southern analysis showing promoter methylation at the Nesp DMR is lost in +/ Nespas-T ex1 . Genomic DNA from newborn brain was digested with Eco RI (-), Eco RI and Hpa II (H) or Eco RI and Msp I (M). (E) Biallelic expression of Nesp in +/ Nespas-T ex1 . (E Top Left) RNA blot analysis showing expression of Nesp in poly(A) + RNA from 15.5 dpc embryos. (E Bottom Left) Schematic summary of the transcriptional and methylation status of Nespas and Nesp on the paternal allele of +/ Nespas-T ex1 . (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR, measured using a TaqMan assay that detects exon 1 spliced onto 2. The reference gene was Gapdh . Error bars (RQmin/RQmax) were based on a 95% confidence level.
    Figure Legend Snippet: Nesp is fully expressed from the paternal allele in +/ Nespas-T ex1 . (A) The Nespas-T ex1 allele was generated by targeting and is identical to Nesp-T int2 except that the polyadenylation cassette was inserted in the opposite orientation (Ap) to truncate Nespas. The selection genes were deleted from the Ap- neo targeted allele upon germline transmission by Cre-recombinase mediated excision to generate Nespas-T ex1 . (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (Ap- neo ) cells. The Ap- neo targeted clones were identified by the presence of an 8.5 kb Nde I fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb Avr II fragment with the 5′ external probe. (C) RT-PCR assay in newborn brain showing Nespas is truncated. A primer pair, 3′ of the insertion, detected Nespas whereas a primer pair 5′ of the insertion did not detect Nespas . Symbols + and −, refer to reactions carried out in the presence and absence of reverse transcriptase, respectively. As the Nespas PCR products were weak, it was necessary to blot and probe the Nespas RT-PCR products with appropriate genomic probes. Hprt was included as an amplification control. (D) Southern analysis showing promoter methylation at the Nesp DMR is lost in +/ Nespas-T ex1 . Genomic DNA from newborn brain was digested with Eco RI (-), Eco RI and Hpa II (H) or Eco RI and Msp I (M). (E) Biallelic expression of Nesp in +/ Nespas-T ex1 . (E Top Left) RNA blot analysis showing expression of Nesp in poly(A) + RNA from 15.5 dpc embryos. (E Bottom Left) Schematic summary of the transcriptional and methylation status of Nespas and Nesp on the paternal allele of +/ Nespas-T ex1 . (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR, measured using a TaqMan assay that detects exon 1 spliced onto 2. The reference gene was Gapdh . Error bars (RQmin/RQmax) were based on a 95% confidence level.

    Techniques Used: Generated, Selection, Transmission Assay, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Methylation, Expressing, Northern blot, Quantitative RT-PCR, TaqMan Assay

    Maternal transmission of Nesp-T int2 results in downregulation of Nesp when the Nespas DMR is unmethylated. (A) The maternal Nesp transcript is truncated in Nesp-T int2 /+. RNA blot analysis showing expression of Nesp in poly(A) + RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C . (B) Southern analysis showing the Nespas - Gnasxl DMR promoter can be unmethylated or methylated on the maternal allele in Nesp-T int2 /+. Genomic DNA from newborn brain was digested with Eco RI (-), Eco RI and Hpa II (H), or Eco RI and Msp I (M) and probed as shown in Figure S1 . (C) Schematic showing the genomic organization of the double heterozygote with Nesp-T int2 maternally inherited and Δ NAS-DMR paternally inherited. The polyadenylation cassette, pA, truncates Nesp on the maternal allele and the ICR is deleted on the paternal allele. (D) RT-PCR showing Nespas is weakly expressed in neonatal brain from the maternally-derived targeted allele when the Nespas DMR is unmethylated. Primers were as described in Figure 2C . + and −, presence and absence of reverse transcriptase, respectively; Hprt , amplification control. (E, Left) Schematic summary of the transcriptional and methylation status of the maternal allele of Nesp and Nespas in Nesp-T int2 /+ (methylated) and Nesp-T int2 /+ (unmethylated) newborn brain. The polyadenylation cassette, pA truncates Nesp on the maternal allele. Row of filled circles, methylated allele; row of open circles, unmethylated allele. (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR between Nesp-T int2 /+ SD2 littermates that had the Nespas DMR methylated and unmethylated. Nesp levels were measured using the TaqMan assay detecting exon 1 spliced onto exon 2. The level of Nesp was significantly lower in littermates that had the Nespas DMR unmethylated compared with those that had a methylated DMR ( P = 5.4×10 −3 ).
    Figure Legend Snippet: Maternal transmission of Nesp-T int2 results in downregulation of Nesp when the Nespas DMR is unmethylated. (A) The maternal Nesp transcript is truncated in Nesp-T int2 /+. RNA blot analysis showing expression of Nesp in poly(A) + RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C . (B) Southern analysis showing the Nespas - Gnasxl DMR promoter can be unmethylated or methylated on the maternal allele in Nesp-T int2 /+. Genomic DNA from newborn brain was digested with Eco RI (-), Eco RI and Hpa II (H), or Eco RI and Msp I (M) and probed as shown in Figure S1 . (C) Schematic showing the genomic organization of the double heterozygote with Nesp-T int2 maternally inherited and Δ NAS-DMR paternally inherited. The polyadenylation cassette, pA, truncates Nesp on the maternal allele and the ICR is deleted on the paternal allele. (D) RT-PCR showing Nespas is weakly expressed in neonatal brain from the maternally-derived targeted allele when the Nespas DMR is unmethylated. Primers were as described in Figure 2C . + and −, presence and absence of reverse transcriptase, respectively; Hprt , amplification control. (E, Left) Schematic summary of the transcriptional and methylation status of the maternal allele of Nesp and Nespas in Nesp-T int2 /+ (methylated) and Nesp-T int2 /+ (unmethylated) newborn brain. The polyadenylation cassette, pA truncates Nesp on the maternal allele. Row of filled circles, methylated allele; row of open circles, unmethylated allele. (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR between Nesp-T int2 /+ SD2 littermates that had the Nespas DMR methylated and unmethylated. Nesp levels were measured using the TaqMan assay detecting exon 1 spliced onto exon 2. The level of Nesp was significantly lower in littermates that had the Nespas DMR unmethylated compared with those that had a methylated DMR ( P = 5.4×10 −3 ).

    Techniques Used: Transmission Assay, Northern blot, Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Amplification, Quantitative RT-PCR, TaqMan Assay

    The targeted allele, Nesp-T int2 , is a Nespas hypomorph when paternally inherited. (A) Overview of the mouse Gnas locus showing enlargement of the Nespas region and the site of insertion of the rabbit β-globin polyadenylation cassette in Nespas exon 1. The targeting vector shows the location of a 1.2 kb fragment (pA) from the rabbit β-globin gene to truncate Nesp , and the selection genes were flanked by loxP sites (open triangles). The selection genes were deleted from the pA- neo targeted allele upon male germline transmission by Cre-recombinase mediated excision to generate Nesp-T int2 . The DMRs are shown by the presence of filled circles on the methylated allele. A, Avr II; N, Nde I; Sp, Spe I; Sw, Swa I; P, Nespas promoter; ICR, Imprinting Control Region, the extent of which is defined by the deletion in the Δ NAS-DMR allele [8] . (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (pA- neo ) cells. The pA- neo targeted clones were identified by the presence of an 8.5 kb Nde I fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb Avr II fragment with the 5′ external probe. (C) Schematic of the Nesp - Nespas region showing overlapping transcription on the sense and antisense strands and the position of RT-PCR primers, a single stranded RNA probe and TaqMan assay for detecting the primary Nespas transcript. (D) RT-PCR analysis of Nespas in neonatal brain after paternal transmission of Nesp-T int2 (+/ Nesp-T int2 ) using primers F6 and R2 (Figure 2C; Table S1 ). +/Δ NAS-DMR , mice with paternal deletion of the ICR [8] ; + and −, presence and absence of reverse transcriptase, respectively; Hprt , amplification control. (E) RNA blot analysis showing expression of Nespas in poly(A) + RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C. (F) Bar chart showing the relative level of Nespas expression in newborn brain by RT-qPCR using a TaqMan assay within intron 4 (Figure 2C). The reference gene was Gapdh . Error bars (RQmin/RQmax) were based on a 95% confidence level.
    Figure Legend Snippet: The targeted allele, Nesp-T int2 , is a Nespas hypomorph when paternally inherited. (A) Overview of the mouse Gnas locus showing enlargement of the Nespas region and the site of insertion of the rabbit β-globin polyadenylation cassette in Nespas exon 1. The targeting vector shows the location of a 1.2 kb fragment (pA) from the rabbit β-globin gene to truncate Nesp , and the selection genes were flanked by loxP sites (open triangles). The selection genes were deleted from the pA- neo targeted allele upon male germline transmission by Cre-recombinase mediated excision to generate Nesp-T int2 . The DMRs are shown by the presence of filled circles on the methylated allele. A, Avr II; N, Nde I; Sp, Spe I; Sw, Swa I; P, Nespas promoter; ICR, Imprinting Control Region, the extent of which is defined by the deletion in the Δ NAS-DMR allele [8] . (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (pA- neo ) cells. The pA- neo targeted clones were identified by the presence of an 8.5 kb Nde I fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb Avr II fragment with the 5′ external probe. (C) Schematic of the Nesp - Nespas region showing overlapping transcription on the sense and antisense strands and the position of RT-PCR primers, a single stranded RNA probe and TaqMan assay for detecting the primary Nespas transcript. (D) RT-PCR analysis of Nespas in neonatal brain after paternal transmission of Nesp-T int2 (+/ Nesp-T int2 ) using primers F6 and R2 (Figure 2C; Table S1 ). +/Δ NAS-DMR , mice with paternal deletion of the ICR [8] ; + and −, presence and absence of reverse transcriptase, respectively; Hprt , amplification control. (E) RNA blot analysis showing expression of Nespas in poly(A) + RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C. (F) Bar chart showing the relative level of Nespas expression in newborn brain by RT-qPCR using a TaqMan assay within intron 4 (Figure 2C). The reference gene was Gapdh . Error bars (RQmin/RQmax) were based on a 95% confidence level.

    Techniques Used: Plasmid Preparation, Selection, Transmission Assay, Methylation, Clone Assay, Reverse Transcription Polymerase Chain Reaction, TaqMan Assay, Mouse Assay, Amplification, Northern blot, Expressing, Quantitative RT-PCR

    17) Product Images from "RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes"

    Article Title: RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00775

    Design and execution of the small-hairpin (sh)RNA screens for murine nuclear cofactors of IL-1 signaling. For screen I, 4–5 shRNAs directed at up to four different nuclear targets per 48-well plate were transfected in duplicates as shown. In screen II, 4–5 shRNAs per nuclear target were pooled and transfected into a single well resulting in screening of 20 nuclear targets per plate (not shown). In both screens, empty vector (pLKO.1) with no insert or an insert encoding a scrambled shRNA sequence (pLKO.1-scr.) were used as controls on each of the plates. Cells transfected with pLKO.1 encoding a GFP cDNA were used to monitor transfection efficiency on each individual plate by fluorescence microscopy (left image) and by phase contrast plus fluorescence microscopy (right image) as shown by the insets. The scale bar is 100 µm. For each screen, 3.5 × 10 4 cells were seeded per well. One day later, 270 ng of DNA were transfected using Lipofectamine LTX plus reagent ® . Cells were selected for 48 h in 1 µg/ml puromycin. Then, half of the cells on each plate were left untreated. The other half was stimulated for 3 h (screen I) or 1 h (screen II) with IL-1α (10 ng/ml). Thereafter, cells were lysed, and RT reactions and preamplifications of cDNAs were performed using the PreAmp Cells-to-Ct TM Kit and gene specific primers for the IL-1-inducible target gene Cxcl2 and the two “housekeeping” genes ActB (screen I only) and Ube2l3 . Finally, preamplified PCR products were subjected to quantitative (q)PCR using Taqman assays and mRNA expressions levels were quantified based on their cycle threshold (ct) value using an ABI7500 instrument. Ct values were used for all further calculations.
    Figure Legend Snippet: Design and execution of the small-hairpin (sh)RNA screens for murine nuclear cofactors of IL-1 signaling. For screen I, 4–5 shRNAs directed at up to four different nuclear targets per 48-well plate were transfected in duplicates as shown. In screen II, 4–5 shRNAs per nuclear target were pooled and transfected into a single well resulting in screening of 20 nuclear targets per plate (not shown). In both screens, empty vector (pLKO.1) with no insert or an insert encoding a scrambled shRNA sequence (pLKO.1-scr.) were used as controls on each of the plates. Cells transfected with pLKO.1 encoding a GFP cDNA were used to monitor transfection efficiency on each individual plate by fluorescence microscopy (left image) and by phase contrast plus fluorescence microscopy (right image) as shown by the insets. The scale bar is 100 µm. For each screen, 3.5 × 10 4 cells were seeded per well. One day later, 270 ng of DNA were transfected using Lipofectamine LTX plus reagent ® . Cells were selected for 48 h in 1 µg/ml puromycin. Then, half of the cells on each plate were left untreated. The other half was stimulated for 3 h (screen I) or 1 h (screen II) with IL-1α (10 ng/ml). Thereafter, cells were lysed, and RT reactions and preamplifications of cDNAs were performed using the PreAmp Cells-to-Ct TM Kit and gene specific primers for the IL-1-inducible target gene Cxcl2 and the two “housekeeping” genes ActB (screen I only) and Ube2l3 . Finally, preamplified PCR products were subjected to quantitative (q)PCR using Taqman assays and mRNA expressions levels were quantified based on their cycle threshold (ct) value using an ABI7500 instrument. Ct values were used for all further calculations.

    Techniques Used: Transfection, Plasmid Preparation, shRNA, Sequencing, Fluorescence, Microscopy, Polymerase Chain Reaction

    18) Product Images from "Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation"

    Article Title: Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2013060675

    Evaluation of M1-like M ϕ -related genes. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the average of the data for the +/+ mice on day 0. The data are presented as means±SEMs. Day 6 (CSF-1) indicates that all mice in each genotype group were administered 5.0 μ g CSF-1. n =6 for each group. * P
    Figure Legend Snippet: Evaluation of M1-like M ϕ -related genes. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the average of the data for the +/+ mice on day 0. The data are presented as means±SEMs. Day 6 (CSF-1) indicates that all mice in each genotype group were administered 5.0 μ g CSF-1. n =6 for each group. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

    Evaluation of crystal binding and related gene expressions. (A) The expression of each gene indicated was determined by quantitative RT-PCR using TaqMan assays. Control values represent the average of the data for the +/+ mice on day 0. The data are presented as means±SEMs. Day 6 (CSF-1) indicates that all mice in each genotype group were treated with 5.0 μg CSF-1. n =6 for each group. * P
    Figure Legend Snippet: Evaluation of crystal binding and related gene expressions. (A) The expression of each gene indicated was determined by quantitative RT-PCR using TaqMan assays. Control values represent the average of the data for the +/+ mice on day 0. The data are presented as means±SEMs. Day 6 (CSF-1) indicates that all mice in each genotype group were treated with 5.0 μg CSF-1. n =6 for each group. * P

    Techniques Used: Binding Assay, Expressing, Quantitative RT-PCR, Mouse Assay

    Evaluation of pan-M ϕ –related gene expression. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the averages of the data for the +/+ mice on day 0. The data are presented as means±SEMs. Day 6 (CSF-1) indicates that all mice in each genotype group were administered 5.0 μ g CSF-1. n =6 for each group. * P
    Figure Legend Snippet: Evaluation of pan-M ϕ –related gene expression. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the averages of the data for the +/+ mice on day 0. The data are presented as means±SEMs. Day 6 (CSF-1) indicates that all mice in each genotype group were administered 5.0 μ g CSF-1. n =6 for each group. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

    Evaluation of M2-like M ϕ -related genes. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the average of the data for the +/+ mice on day 0. The data represent mean±SEM values. Day 6 (CSF-1) indicates that every mouse in each genotype group was administered 5.0 μ g CSF-1. n =6 for each group. * P
    Figure Legend Snippet: Evaluation of M2-like M ϕ -related genes. (A) The expression of each gene was determined by quantitative RT-PCR using TaqMan assays. Control values are the average of the data for the +/+ mice on day 0. The data represent mean±SEM values. Day 6 (CSF-1) indicates that every mouse in each genotype group was administered 5.0 μ g CSF-1. n =6 for each group. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

    19) Product Images from "Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes"

    Article Title: Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl416

    The C T value as a function of annealing/elongation time is shown in red. This demonstrated that the Taqman PCR system can be run with an annealing/elongation step of 10 s. Even with this step as short as 6.5 s, the PCR performance is not significantly affected. This PCR system is then capable of running a complete 50 cycle PCR protocol in 7 min and 25 s inclusive of a 20 s hot start.
    Figure Legend Snippet: The C T value as a function of annealing/elongation time is shown in red. This demonstrated that the Taqman PCR system can be run with an annealing/elongation step of 10 s. Even with this step as short as 6.5 s, the PCR performance is not significantly affected. This PCR system is then capable of running a complete 50 cycle PCR protocol in 7 min and 25 s inclusive of a 20 s hot start.

    Techniques Used: Polymerase Chain Reaction

    20) Product Images from "Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13"

    Article Title: Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101270

    30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) Taqman PCR assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p
    Figure Legend Snippet: 30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) Taqman PCR assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p

    Techniques Used: Expressing, Microarray, Hybridization, Polymerase Chain Reaction, Mouse Assay

    21) Product Images from "Region-Specific Differences in Amyloid Precursor Protein Expression in the Mouse Hippocampus"

    Article Title: Region-Specific Differences in Amyloid Precursor Protein Expression in the Mouse Hippocampus

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2016.00134

    Quantitative App mRNA expression in hippocampal subregions. (A,B) LMD of hippocampal subregions, i.e., CA1 pcl and gcl of the DG. Representative section of the dorsal hippocampus (coronal plane, cresyl violet staining) before (A) and after (B) LMD is shown. Scale bar: 250 μm. (C) RNA integrity analysis of total RNA isolated from the dissected gcl (red) and from pcl (blue) demonstrating highly intact RNA (RIN-values: ~9; Agilent 2100 Bioanalyzer). (D,E) Average expression stability values ( M ) and evaluation of the optimum number of candidate reference genes for CA1 pcl and for DG gcl according to geNorm software. Pairwise variation (V) of candidate reference genes indicates that the use of the two most stable genes is sufficient to obtain an accurate normalization index for quantitative PCR (qPCR) analysis. (F) A significantly higher gene expression for App (1.5- to 1.7-fold) was detected in CA1 pcl relative to DG gcl using two different App -specific TaqMan assays ( A : Mm_01344172_m1, B : Mm_00431830_m1) after normalization to a reference gene index calculated by geNorm. (G,H) Gene expression stability values ( S ) and accumulated SD analysis using NormFinder. The minimal number of reference genes required for effective normalization is highlighted. (I) Comparable to the results obtained by geNorm algorithm, a significantly higher App expression (1.6- to 1.7-fold) was detected in CA1 pcl relative to DG gcl after normalization to the reference gene index estimated by NormFinder. Data ( N = 5–6 mice) were tested for statistical significance using one-way ANOVA followed by Bonferroni post hoc test to correct for multiple comparisons, * p ≤ 0.05.
    Figure Legend Snippet: Quantitative App mRNA expression in hippocampal subregions. (A,B) LMD of hippocampal subregions, i.e., CA1 pcl and gcl of the DG. Representative section of the dorsal hippocampus (coronal plane, cresyl violet staining) before (A) and after (B) LMD is shown. Scale bar: 250 μm. (C) RNA integrity analysis of total RNA isolated from the dissected gcl (red) and from pcl (blue) demonstrating highly intact RNA (RIN-values: ~9; Agilent 2100 Bioanalyzer). (D,E) Average expression stability values ( M ) and evaluation of the optimum number of candidate reference genes for CA1 pcl and for DG gcl according to geNorm software. Pairwise variation (V) of candidate reference genes indicates that the use of the two most stable genes is sufficient to obtain an accurate normalization index for quantitative PCR (qPCR) analysis. (F) A significantly higher gene expression for App (1.5- to 1.7-fold) was detected in CA1 pcl relative to DG gcl using two different App -specific TaqMan assays ( A : Mm_01344172_m1, B : Mm_00431830_m1) after normalization to a reference gene index calculated by geNorm. (G,H) Gene expression stability values ( S ) and accumulated SD analysis using NormFinder. The minimal number of reference genes required for effective normalization is highlighted. (I) Comparable to the results obtained by geNorm algorithm, a significantly higher App expression (1.6- to 1.7-fold) was detected in CA1 pcl relative to DG gcl after normalization to the reference gene index estimated by NormFinder. Data ( N = 5–6 mice) were tested for statistical significance using one-way ANOVA followed by Bonferroni post hoc test to correct for multiple comparisons, * p ≤ 0.05.

    Techniques Used: Expressing, Laser Capture Microdissection, Staining, Isolation, Software, Real-time Polymerase Chain Reaction, Mouse Assay

    22) Product Images from "Optimized Surface Markers for the Prospective Isolation of High-Quality hiPSCs using Flow Cytometry Selection"

    Article Title: Optimized Surface Markers for the Prospective Isolation of High-Quality hiPSCs using Flow Cytometry Selection

    Journal: Scientific Reports

    doi: 10.1038/srep01179

    hiPSC selection by sorting SSEA4 + TRA-1-81 + CD30 + cells yields pluripotent feeder-free hiPSC lines with stable genome and tri-lineage differentiation potential. Four weeks post initiation of reprogramming of FTc91 human fibroblasts, hiPSCs were separated by FACS and using the SSEA4, TRA-1-81, and CD30 markers as described above. Three iPSC lines FTi115, FTi116, and FTi117 were established and characterized. (a) Flow cytometry analyses of indicated hiPSCs. (b) Indicated iPSC lines were immunostained for expression of NANOG (green) and OCT4 (red). Nuclei were stained with Hoechst dye (blue). Scale bar is 200 μm. (c) Nanog+ and Oct4+ FTi117 iPSCs were quanitified by intracellular flow cytometry. (d), (e) qRT-PCR analysis of gene expression of pluripotency markers (d) and transgene (e) in indicated hiPSC lines. The lentivirus used for reprogramming expressed the transgenes as a single polycistronic cassette. Transgene levels were measured by a TaqMan primer-probe set within the viral WPRE element. Error bars represent standard deviation of duplicates. The asterisks denote corresponding p-values (*
    Figure Legend Snippet: hiPSC selection by sorting SSEA4 + TRA-1-81 + CD30 + cells yields pluripotent feeder-free hiPSC lines with stable genome and tri-lineage differentiation potential. Four weeks post initiation of reprogramming of FTc91 human fibroblasts, hiPSCs were separated by FACS and using the SSEA4, TRA-1-81, and CD30 markers as described above. Three iPSC lines FTi115, FTi116, and FTi117 were established and characterized. (a) Flow cytometry analyses of indicated hiPSCs. (b) Indicated iPSC lines were immunostained for expression of NANOG (green) and OCT4 (red). Nuclei were stained with Hoechst dye (blue). Scale bar is 200 μm. (c) Nanog+ and Oct4+ FTi117 iPSCs were quanitified by intracellular flow cytometry. (d), (e) qRT-PCR analysis of gene expression of pluripotency markers (d) and transgene (e) in indicated hiPSC lines. The lentivirus used for reprogramming expressed the transgenes as a single polycistronic cassette. Transgene levels were measured by a TaqMan primer-probe set within the viral WPRE element. Error bars represent standard deviation of duplicates. The asterisks denote corresponding p-values (*

    Techniques Used: Selection, FACS, Flow Cytometry, Cytometry, Expressing, Staining, Quantitative RT-PCR, Standard Deviation

    23) Product Images from "BANK1 Controls CpG-induced IL-6 Secretion Via a p38 and MNK1/2- eIF4E Translation Initiation Pathway"

    Article Title: BANK1 Controls CpG-induced IL-6 Secretion Via a p38 and MNK1/2- eIF4E Translation Initiation Pathway

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1301203

    CpG-induced IL-6 production is downregulated in Bank1−/− B cells, but Tlr9 gene expression is not affected (A) IL-6 and IL-10 were measured by ELISA of WT ( +/+ ) and Bank1 KO ( −/− ) B-cell supernatants collected at the indicated times following CpG, anti-IgM + CpG or CpG + anti-mouse CD40 (10μg/ml). Data shown are mean ± SD of 3 replicates, and is representative of 4 independent experiments. (B) Splenic B cells from Bank1 +/+ and Bank1 −/− mice were stimulated with 2μM CpG for the indicated times. Relative expression of the Tlr9 gene was analyzed by Taqman real-time PCR. Data shown is the mean ± SEM of 3 replicates from a representative experiment out of 3 performed. (C) Bank1 +/+ and Bank1 −/− splenic B cells were stimulated with LPS (20 μg/ml) or R848 (1 μg/mL), or left unstimulated for the times shown. Data are the representative of two independent experiments with 3 replicates each. Bars represent mean ± SD.
    Figure Legend Snippet: CpG-induced IL-6 production is downregulated in Bank1−/− B cells, but Tlr9 gene expression is not affected (A) IL-6 and IL-10 were measured by ELISA of WT ( +/+ ) and Bank1 KO ( −/− ) B-cell supernatants collected at the indicated times following CpG, anti-IgM + CpG or CpG + anti-mouse CD40 (10μg/ml). Data shown are mean ± SD of 3 replicates, and is representative of 4 independent experiments. (B) Splenic B cells from Bank1 +/+ and Bank1 −/− mice were stimulated with 2μM CpG for the indicated times. Relative expression of the Tlr9 gene was analyzed by Taqman real-time PCR. Data shown is the mean ± SEM of 3 replicates from a representative experiment out of 3 performed. (C) Bank1 +/+ and Bank1 −/− splenic B cells were stimulated with LPS (20 μg/ml) or R848 (1 μg/mL), or left unstimulated for the times shown. Data are the representative of two independent experiments with 3 replicates each. Bars represent mean ± SD.

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

    Reduced IL-6 secretion can be attributed to downregulation of the phosphorylation of the p38-MNK1/2-eIF4E cascade in Bank1 −/− B cells following CpG stimulation and not to Il6 gene transcription and Il6 mRNA stability (A) Splenic B cells from Bank1+/+ and Bank1−/− mice were stimulated with 2 μM CpG for indicated time shown on the plot. Relative expression of the Il6 gene was determined using Taqman RT-PCR. Data shown is the mean ± SEM of 3 replicates from 1 out of 3 independent experiments. (B) Splenic B cells from Bank1+/+ and Bank1−/− littermate mice were stimulated with 2μM CpG for 20h followed by the addition of 1μg/mL of actinomycin D (Act D). Relative expression of the Il6 gene was analyzed with RT-PCR, and % of Il6 mRNA was determined. Data shown is the mean of 3 replicates from 1 representative experiment out of 3 performed. The thin dotted line indicates 50% degradation of Il6 mRNA. (C) Bank1 +/+ or −/− splenic B cells were stimulated with 2μM CpG for the indicated times and tested by Western blot using p-MNK1/2, MNK1/2, p-eIF4E, and eIF4E specific antibodies. The data are representative of 3 independent experiments. (D) Quantification analysis of phospho-MNK1/2 and phospho-eIF4E Western blot data by densitometry. The relative intensities of phospho-MNK1/2 and phospho-eIF4E were quantified using ImageJ software (freely available through the National Institutes of Health). The plots are from 3 independent experiments and data are expressed as mean ± SD.
    Figure Legend Snippet: Reduced IL-6 secretion can be attributed to downregulation of the phosphorylation of the p38-MNK1/2-eIF4E cascade in Bank1 −/− B cells following CpG stimulation and not to Il6 gene transcription and Il6 mRNA stability (A) Splenic B cells from Bank1+/+ and Bank1−/− mice were stimulated with 2 μM CpG for indicated time shown on the plot. Relative expression of the Il6 gene was determined using Taqman RT-PCR. Data shown is the mean ± SEM of 3 replicates from 1 out of 3 independent experiments. (B) Splenic B cells from Bank1+/+ and Bank1−/− littermate mice were stimulated with 2μM CpG for 20h followed by the addition of 1μg/mL of actinomycin D (Act D). Relative expression of the Il6 gene was analyzed with RT-PCR, and % of Il6 mRNA was determined. Data shown is the mean of 3 replicates from 1 representative experiment out of 3 performed. The thin dotted line indicates 50% degradation of Il6 mRNA. (C) Bank1 +/+ or −/− splenic B cells were stimulated with 2μM CpG for the indicated times and tested by Western blot using p-MNK1/2, MNK1/2, p-eIF4E, and eIF4E specific antibodies. The data are representative of 3 independent experiments. (D) Quantification analysis of phospho-MNK1/2 and phospho-eIF4E Western blot data by densitometry. The relative intensities of phospho-MNK1/2 and phospho-eIF4E were quantified using ImageJ software (freely available through the National Institutes of Health). The plots are from 3 independent experiments and data are expressed as mean ± SD.

    Techniques Used: Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Activated Clotting Time Assay, Western Blot, Software

    24) Product Images from "Dynamics of molecular responses to coxsackievirus B4 infection differentiate between resolution and progression of acute pancreatitis"

    Article Title: Dynamics of molecular responses to coxsackievirus B4 infection differentiate between resolution and progression of acute pancreatitis

    Journal: Virology

    doi: 10.1016/j.virol.2012.02.009

    Relative change in expression profiles of functional groupings of genes after CVB4-V infection of BALB/c and IL-10 KO mice. A. TLR related grouping; B. A miscellaneous grouping consisting of three genes, Gata3, Socs3, and Il10. Three-to-five mice per strain were analyzed at each time point and gene expression in the pancreas was quantified using the TaqMan Gene Expression Assay in a two-step RT-PCR and a low-density array. The Ct value of each test gene was normalized to an endogenous control, egf, and the [delta] Ct values for samples at each time point were averaged. The change in gene expression, for both BALB/c and IL-10 KO mice, is represented as a fold-change relative to the expression level of a reference group which is BALB/c mice at 2dpi. As a result, the relative expression of a gene in BALB/c mice at 2dpi is 1. Closed circles, BALB/c mice; open circles, IL-10 KO mice.
    Figure Legend Snippet: Relative change in expression profiles of functional groupings of genes after CVB4-V infection of BALB/c and IL-10 KO mice. A. TLR related grouping; B. A miscellaneous grouping consisting of three genes, Gata3, Socs3, and Il10. Three-to-five mice per strain were analyzed at each time point and gene expression in the pancreas was quantified using the TaqMan Gene Expression Assay in a two-step RT-PCR and a low-density array. The Ct value of each test gene was normalized to an endogenous control, egf, and the [delta] Ct values for samples at each time point were averaged. The change in gene expression, for both BALB/c and IL-10 KO mice, is represented as a fold-change relative to the expression level of a reference group which is BALB/c mice at 2dpi. As a result, the relative expression of a gene in BALB/c mice at 2dpi is 1. Closed circles, BALB/c mice; open circles, IL-10 KO mice.

    Techniques Used: Expressing, Functional Assay, Infection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    Relative change in expression profiles of TH17 related genes after CVB4-V infection of BALB/c and IL-10 KO mice. Three-to-five mice per strain were analyzed at each time point and gene expression in the pancreas was quantified using the TaqMan Gene Expression Assay in a two-step RT-PCR and a low-density array. The Ct value of each test gene was normalized to an endogenous control, egf, and the [delta] Ct values for samples at each time point were averaged. The change in gene expression is represented as a fold-change relative to the expression level of a reference group. For all genes except RORγt, the reference group is BALB/c at 2dpi. For RORγt, the reference group is BALB/c at 4 dpi. As a result, the relative expression of a gene, except for RORγt, in BALB/c mice at 2dpi is 1. The relative expression of RORγt in BALB/c mice at 4dpi is 1. Closed circles, BALB/c mice; open circles, IL-10 KO mice.
    Figure Legend Snippet: Relative change in expression profiles of TH17 related genes after CVB4-V infection of BALB/c and IL-10 KO mice. Three-to-five mice per strain were analyzed at each time point and gene expression in the pancreas was quantified using the TaqMan Gene Expression Assay in a two-step RT-PCR and a low-density array. The Ct value of each test gene was normalized to an endogenous control, egf, and the [delta] Ct values for samples at each time point were averaged. The change in gene expression is represented as a fold-change relative to the expression level of a reference group. For all genes except RORγt, the reference group is BALB/c at 2dpi. For RORγt, the reference group is BALB/c at 4 dpi. As a result, the relative expression of a gene, except for RORγt, in BALB/c mice at 2dpi is 1. The relative expression of RORγt in BALB/c mice at 4dpi is 1. Closed circles, BALB/c mice; open circles, IL-10 KO mice.

    Techniques Used: Expressing, Infection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    25) Product Images from "A molecular trigger for intercontinental epidemics of group A Streptococcus"

    Article Title: A molecular trigger for intercontinental epidemics of group A Streptococcus

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI82478

    An isoallelic mutant GAS strain that overexpresses NADase and SLO is significantly more virulent than MGAS2221 in a mouse model of necrotizing fasciitis. ( A ) Transcript levels of nga and slo in MGAS2221, 2-SNP mutant nga (G-22T/T-18C), Nga/Slo-OE (overexpressing), and Nga/Slo-DE (2-nucleotide deletion) as determined by TaqMan qRT-PCR ( n = 3 per strain). Data are expressed as the mean ± SD. * P
    Figure Legend Snippet: An isoallelic mutant GAS strain that overexpresses NADase and SLO is significantly more virulent than MGAS2221 in a mouse model of necrotizing fasciitis. ( A ) Transcript levels of nga and slo in MGAS2221, 2-SNP mutant nga (G-22T/T-18C), Nga/Slo-OE (overexpressing), and Nga/Slo-DE (2-nucleotide deletion) as determined by TaqMan qRT-PCR ( n = 3 per strain). Data are expressed as the mean ± SD. * P

    Techniques Used: Mutagenesis, Quantitative RT-PCR

    An analogous molecular event has triggered an intercontinental epidemic of infections caused by serotype M89 GAS. ( A ) Epidemic curve showing increase in frequency of infections caused by progeny of a new clone (variant 3) of serotype M89 strains. Data are from comprehensive population-based studies in the US, Finland, and Iceland. ( B ) Three nga promoter variants in the M89 strains. The genetically representative strains used ( C ) have variant 1 (MGAS11027), variant 2 (MGAS23530), and variant 3 (MGAS26844) patterns. ( C ) TaqMan qRT-PCR analysis of in vivo nga and slo transcript levels in the 3 strains. Transcript levels are displayed relative to the strain MGAS26844 (with variant 3) transcript level. Cultures were analyzed in triplicate on 3 separate occasions. NADase and SLO activity data for wild-type strain MGAS26844 (variant 3) and isogenic mutant strains with variant 2 (26844-V2) and variant 1 (26844-V1) nga promoter. Transcript data and NADase and SLO activity data are shown as mean ± SD ( n = 3). Statistical significance was determined by 2-tailed Student’s t test. ( D ) Mouse survival data. Shown are Kaplan-Meier survival curves. Mice ( n = 15 per strain) were inoculated in the hind limb with epidemic parental wild-type strain MGAS26844 or isoallelic mutant strains. P values were determined using the log-rank test.
    Figure Legend Snippet: An analogous molecular event has triggered an intercontinental epidemic of infections caused by serotype M89 GAS. ( A ) Epidemic curve showing increase in frequency of infections caused by progeny of a new clone (variant 3) of serotype M89 strains. Data are from comprehensive population-based studies in the US, Finland, and Iceland. ( B ) Three nga promoter variants in the M89 strains. The genetically representative strains used ( C ) have variant 1 (MGAS11027), variant 2 (MGAS23530), and variant 3 (MGAS26844) patterns. ( C ) TaqMan qRT-PCR analysis of in vivo nga and slo transcript levels in the 3 strains. Transcript levels are displayed relative to the strain MGAS26844 (with variant 3) transcript level. Cultures were analyzed in triplicate on 3 separate occasions. NADase and SLO activity data for wild-type strain MGAS26844 (variant 3) and isogenic mutant strains with variant 2 (26844-V2) and variant 1 (26844-V1) nga promoter. Transcript data and NADase and SLO activity data are shown as mean ± SD ( n = 3). Statistical significance was determined by 2-tailed Student’s t test. ( D ) Mouse survival data. Shown are Kaplan-Meier survival curves. Mice ( n = 15 per strain) were inoculated in the hind limb with epidemic parental wild-type strain MGAS26844 or isoallelic mutant strains. P values were determined using the log-rank test.

    Techniques Used: Variant Assay, Quantitative RT-PCR, In Vivo, Activity Assay, Mutagenesis, Mouse Assay

    In vitro characteristics of GAS M1 reference and isoallelic mutant strains. ( A ) Schematic showing the target SNP genotype of the strains assayed. ( B ) TaqMan qRT-PCR analysis of nga and slo transcript levels relative to MGAS2221 levels. ( C ) Western immunoblot analysis of secreted SPN and SLO in culture supernatants. ( D ) SLO cytolytic activity relative to MGAS2221 and SPN NADase activity. Transcript, cytolysis, and NADase assays were performed in triplicate on 3 separate occasions. Replicate data are expressed as the mean ± SD in B and D . * P
    Figure Legend Snippet: In vitro characteristics of GAS M1 reference and isoallelic mutant strains. ( A ) Schematic showing the target SNP genotype of the strains assayed. ( B ) TaqMan qRT-PCR analysis of nga and slo transcript levels relative to MGAS2221 levels. ( C ) Western immunoblot analysis of secreted SPN and SLO in culture supernatants. ( D ) SLO cytolytic activity relative to MGAS2221 and SPN NADase activity. Transcript, cytolysis, and NADase assays were performed in triplicate on 3 separate occasions. Replicate data are expressed as the mean ± SD in B and D . * P

    Techniques Used: In Vitro, Mutagenesis, Quantitative RT-PCR, Western Blot, Activity Assay

    26) Product Images from "Photoactivated inhibition of cathepsin K in a 3D tumor model"

    Article Title: Photoactivated inhibition of cathepsin K in a 3D tumor model

    Journal: Biological chemistry

    doi: 10.1515/hsz-2015-0274

    Generation of CK (CTSK) overexpressing human prostate tumor cells (A) Taqman RT-PCR analysis of human CK gene expression in CK-1 and CK-2 clones. Data are normalized to HPRT1 and shown as average fold increase relative to EV (for 3 replicate experiments). (B) Western blot analysis of CK protein (37 kDa proenzyme and 28 kDa mature enzyme; top panel) and DDK (middle panel) expression in representative samples from PC3 prostate carcinoma cells stably transfected with empty vector (PC3-EV) or CTSK plasmids with DDK tag (PC3-CTSK); tubulin was used as loading control. (C) Immunofluorescent staining for DDK (green, left panel) indicative of CTSK expression; no primary antibody staining is shown as negative control (right panel); DAPI (blue) indicates nuclei; 40 × original magnification. (D) CK activity in CK clones. Assay was run against CK substrate Z-Gly-Pro-Arg-AMC the presence of cathepsin B inhibitor CA074. Data are shown as fold increase relative to CTSK activity in PC3-EV cells and a representative of three independent experiments. ** p
    Figure Legend Snippet: Generation of CK (CTSK) overexpressing human prostate tumor cells (A) Taqman RT-PCR analysis of human CK gene expression in CK-1 and CK-2 clones. Data are normalized to HPRT1 and shown as average fold increase relative to EV (for 3 replicate experiments). (B) Western blot analysis of CK protein (37 kDa proenzyme and 28 kDa mature enzyme; top panel) and DDK (middle panel) expression in representative samples from PC3 prostate carcinoma cells stably transfected with empty vector (PC3-EV) or CTSK plasmids with DDK tag (PC3-CTSK); tubulin was used as loading control. (C) Immunofluorescent staining for DDK (green, left panel) indicative of CTSK expression; no primary antibody staining is shown as negative control (right panel); DAPI (blue) indicates nuclei; 40 × original magnification. (D) CK activity in CK clones. Assay was run against CK substrate Z-Gly-Pro-Arg-AMC the presence of cathepsin B inhibitor CA074. Data are shown as fold increase relative to CTSK activity in PC3-EV cells and a representative of three independent experiments. ** p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Clone Assay, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Staining, Negative Control, Activity Assay

    27) Product Images from "SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1"

    Article Title: SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.00426-16

    PIAS1 repression of ICP0-null mutant HSV-1 replication is additive to that of PML. (A) Bar graph showing the average relative levels of PML or PIAS1 mRNA in transgenic HFt cells that express shRNA against PML (shPML), PIAS1 (shPIAS1), or a nontargeted control (shCtrl). PML or PIAS1 mRNA levels were determined using the TaqMan system of quantitative RT-PCR. Values normalized to GAPDH expression using the threshold cycle (ΔΔ C T ) method are expressed relative to mock-infected cells (1.0). Means and standard deviations (SD) are shown ( n > 3). (B) Western blots show PML or PIAS1 protein levels in transgenic HFt cells that express shPML, shPIAS1, or shCtrl. Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PML or PIAS1 and for actin as a loading control. Molecular masses are indicated. (C) Bar graph showing the average relative plaque forming efficiency (PFE) of wild-type (HSV-1) or ICP0-null mutant (ΔICP0) HSV-1 in transgenic HFt cells that express shCtrl, shPML, or shPIAS1. The number of plaques for each strain is expressed relative to the corresponding number of plaques for that strain in shCtrl-expressing cells. Means and SD are shown ( n = 6). Neo, neomycin; Puro, puromycin.
    Figure Legend Snippet: PIAS1 repression of ICP0-null mutant HSV-1 replication is additive to that of PML. (A) Bar graph showing the average relative levels of PML or PIAS1 mRNA in transgenic HFt cells that express shRNA against PML (shPML), PIAS1 (shPIAS1), or a nontargeted control (shCtrl). PML or PIAS1 mRNA levels were determined using the TaqMan system of quantitative RT-PCR. Values normalized to GAPDH expression using the threshold cycle (ΔΔ C T ) method are expressed relative to mock-infected cells (1.0). Means and standard deviations (SD) are shown ( n > 3). (B) Western blots show PML or PIAS1 protein levels in transgenic HFt cells that express shPML, shPIAS1, or shCtrl. Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PML or PIAS1 and for actin as a loading control. Molecular masses are indicated. (C) Bar graph showing the average relative plaque forming efficiency (PFE) of wild-type (HSV-1) or ICP0-null mutant (ΔICP0) HSV-1 in transgenic HFt cells that express shCtrl, shPML, or shPIAS1. The number of plaques for each strain is expressed relative to the corresponding number of plaques for that strain in shCtrl-expressing cells. Means and SD are shown ( n = 6). Neo, neomycin; Puro, puromycin.

    Techniques Used: Mutagenesis, Transgenic Assay, shRNA, Quantitative RT-PCR, Expressing, Infection, Western Blot, SDS Page

    28) Product Images from "Bone marrow adipocytes promote the Warburg phenotype in metastatic prostate tumors via HIF-1α activation"

    Article Title: Bone marrow adipocytes promote the Warburg phenotype in metastatic prostate tumors via HIF-1α activation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11712

    Bone marrow adiposity enhances HIF-1α signaling in PCa cells in vivo prostate bone tumors Taqman RT-PCR analysis of CA9 and VEGF in PC3 cells grown intratibially A. and subcutaneously B. in LFD and HFD fed mice. Data are normalized to EPCAM and shown relative to LFD tumors. C. - G. Immunohistochemical (NovaRED) staining for CA9 protein in prostate bone tumors from mice on LFD C. and HFD E. , 10x images D. , F. High magnification (40x) images depicting membrane CA9 localization in HFD F. but not LFD D. tumors. G. No primary antibody control. Bar, 100μm.
    Figure Legend Snippet: Bone marrow adiposity enhances HIF-1α signaling in PCa cells in vivo prostate bone tumors Taqman RT-PCR analysis of CA9 and VEGF in PC3 cells grown intratibially A. and subcutaneously B. in LFD and HFD fed mice. Data are normalized to EPCAM and shown relative to LFD tumors. C. - G. Immunohistochemical (NovaRED) staining for CA9 protein in prostate bone tumors from mice on LFD C. and HFD E. , 10x images D. , F. High magnification (40x) images depicting membrane CA9 localization in HFD F. but not LFD D. tumors. G. No primary antibody control. Bar, 100μm.

    Techniques Used: In Vivo, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Immunohistochemistry, Staining

    siRNA-mediated knockdown of HIF-1α abrogates bone marrow adipocyte-induced Warburg phenotype in PC3 cells A. Taqman RT-PCR analysis of CA9 , ENO2 , LDHa , HK2 , PDK1 , GLUT1 , and VEGF in PC3 cells cultured in normoxia (20% O 2 ) or hypoxia (1% O 2 ) B. mRNA expression of CA9 , ENO2 , LDHa , HK2 , PDK1 , GLUT1 , and VEGF in response to treatment with 150 μM CoCl 2. C. mRNA levels of HIF-1α in PC3 cells grown under control conditions or treated with 20 μM scrambled siRNA, or 20 μM HIF-1α siRNA. D. Taqman RT-PCR analysis of the expression of HIF-1α target gene CA9 to further confirm HIF-1α knockdown from cells grown in the presence or absence of adipocytes. E-G: Effect of HIF-1α knockdown on the mRNA expression of glycolysis associated genes: PDK1 E. , LDHA F. , and ENO2 G. Data are the mean of analyses with 2 different siRNA constructs done in triplicate. Values * P
    Figure Legend Snippet: siRNA-mediated knockdown of HIF-1α abrogates bone marrow adipocyte-induced Warburg phenotype in PC3 cells A. Taqman RT-PCR analysis of CA9 , ENO2 , LDHa , HK2 , PDK1 , GLUT1 , and VEGF in PC3 cells cultured in normoxia (20% O 2 ) or hypoxia (1% O 2 ) B. mRNA expression of CA9 , ENO2 , LDHa , HK2 , PDK1 , GLUT1 , and VEGF in response to treatment with 150 μM CoCl 2. C. mRNA levels of HIF-1α in PC3 cells grown under control conditions or treated with 20 μM scrambled siRNA, or 20 μM HIF-1α siRNA. D. Taqman RT-PCR analysis of the expression of HIF-1α target gene CA9 to further confirm HIF-1α knockdown from cells grown in the presence or absence of adipocytes. E-G: Effect of HIF-1α knockdown on the mRNA expression of glycolysis associated genes: PDK1 E. , LDHA F. , and ENO2 G. Data are the mean of analyses with 2 different siRNA constructs done in triplicate. Values * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Expressing, Construct

    Bone marrow adipocytes enhance a glycolytic phenotype of prostate cancer cells in direct co-culture and in transwell co-culture in vitro. A. Schematic representation of a direct co-culture of tumor cells and bone marrow adipocytes. B. Taqman RT-PCR analysis of ENO2, LDHa, PDK1, HK2 , and GLUT1 expression in PC3 (Top) and ARCaP(M) (Bottom) cells cultured directly with bone marrow adipocytes. Data are normalized to HPRT1 and shown relative to control. C. Schematic representation of transwell co-cultures of tumor cells and bone marrow adipocytes. D. Taqman RT-PCR of ENO2, LDHa, PDK1, HK2 , and GLUT1 expression in PC3 (top) and ARCaP(M) (bottom) in transwell co-culture. E. Western blot for ENO2, LDHa, HK2, phospho-PDH, and PDK1 in PC3 (left) and ARCaP(M) (right) exposed to bone marrow adipocytes in transwell co-culture. Beta-actin was used as loading control. F. Analysis of lactate secreted (Abcam) by PC3 (left) and ARCaP(M) (right) cells exposed to bone marrow adipocytes in transwell co-culture. Results represent a mean of at least 3 independent experiments ± SD. Values * P
    Figure Legend Snippet: Bone marrow adipocytes enhance a glycolytic phenotype of prostate cancer cells in direct co-culture and in transwell co-culture in vitro. A. Schematic representation of a direct co-culture of tumor cells and bone marrow adipocytes. B. Taqman RT-PCR analysis of ENO2, LDHa, PDK1, HK2 , and GLUT1 expression in PC3 (Top) and ARCaP(M) (Bottom) cells cultured directly with bone marrow adipocytes. Data are normalized to HPRT1 and shown relative to control. C. Schematic representation of transwell co-cultures of tumor cells and bone marrow adipocytes. D. Taqman RT-PCR of ENO2, LDHa, PDK1, HK2 , and GLUT1 expression in PC3 (top) and ARCaP(M) (bottom) in transwell co-culture. E. Western blot for ENO2, LDHa, HK2, phospho-PDH, and PDK1 in PC3 (left) and ARCaP(M) (right) exposed to bone marrow adipocytes in transwell co-culture. Beta-actin was used as loading control. F. Analysis of lactate secreted (Abcam) by PC3 (left) and ARCaP(M) (right) cells exposed to bone marrow adipocytes in transwell co-culture. Results represent a mean of at least 3 independent experiments ± SD. Values * P

    Techniques Used: Co-Culture Assay, In Vitro, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Western Blot

    Paracrine signaling between PCa cells and bone marrow adipocytes is required for the induction of glycolytic gene and protein expression in PCa cells A. Schematic representation of tumor cells treated with media conditioned by bone marrow adipocytes (Adipo CM). B. Taqman RT-PCR analysis of ENO2, LDHa, PDK1, HK2 , and GLUT1 in PC3 (top) and ARCaP(M) (bottom) cells in the presence or absence of Adipo CM. Data are normalized to HPRT1 and shown as increase relative to control. C. Schematic representation of tumor cell- adipocyte co-culture system (CCM). D. Taqman RT PCR analysis of mRNA expression of ENO2, LDHa, PDK1, HK2 , and GLUT1 in PC3 (top) and ARCaP(M) (bottom) cells in the presence of CCM. E. Western blot analysis of ENO2, LDHa, HK2, and phospho-PDH in PC3 (left) and ARCaP(M) (right) in the presence of CCM. Beta-actin was used as a loading control (bottom). Results represent a mean of at least 3 independent experiments ± SD. Values * P
    Figure Legend Snippet: Paracrine signaling between PCa cells and bone marrow adipocytes is required for the induction of glycolytic gene and protein expression in PCa cells A. Schematic representation of tumor cells treated with media conditioned by bone marrow adipocytes (Adipo CM). B. Taqman RT-PCR analysis of ENO2, LDHa, PDK1, HK2 , and GLUT1 in PC3 (top) and ARCaP(M) (bottom) cells in the presence or absence of Adipo CM. Data are normalized to HPRT1 and shown as increase relative to control. C. Schematic representation of tumor cell- adipocyte co-culture system (CCM). D. Taqman RT PCR analysis of mRNA expression of ENO2, LDHa, PDK1, HK2 , and GLUT1 in PC3 (top) and ARCaP(M) (bottom) cells in the presence of CCM. E. Western blot analysis of ENO2, LDHa, HK2, and phospho-PDH in PC3 (left) and ARCaP(M) (right) in the presence of CCM. Beta-actin was used as a loading control (bottom). Results represent a mean of at least 3 independent experiments ± SD. Values * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Co-Culture Assay, Western Blot

    Decreased oxidative phosphorylation in prostate cancer cells exposed to bone marrow adipocyte-derived factors A. Seahorse XF e24 analyzer (Seahorse Bioscience) analysis of the oxygen consumption rate (OCR) in PC3 (top) and ARCaP(M) (bottom) cells upon 12- and 24-hour incubation in the absence or presence of CCM. B. Mitochondrial membrane potential measured via JC-1 fluorescence. C. Taqman RT-PCR analysis of oxidative phosphorylation genes isocitrate dehydrogenase 2 (IDH2) and citrate synthase (CS) after 12 and 24 hours in culture in the absence or presence of CCM. Data are normalized to HPRT1 and shown relative to control. D. ATP levels in PC3 (top) and ARCaP(M) (bottom) cells cultured in the absence or presence of CCM. Significant decrease in ATP levels was observed after 12 hours. CCM exposure had no effect on viability as shown by Calcein AM assay E. and JC-1 apoptosis analyses F. Values * P
    Figure Legend Snippet: Decreased oxidative phosphorylation in prostate cancer cells exposed to bone marrow adipocyte-derived factors A. Seahorse XF e24 analyzer (Seahorse Bioscience) analysis of the oxygen consumption rate (OCR) in PC3 (top) and ARCaP(M) (bottom) cells upon 12- and 24-hour incubation in the absence or presence of CCM. B. Mitochondrial membrane potential measured via JC-1 fluorescence. C. Taqman RT-PCR analysis of oxidative phosphorylation genes isocitrate dehydrogenase 2 (IDH2) and citrate synthase (CS) after 12 and 24 hours in culture in the absence or presence of CCM. Data are normalized to HPRT1 and shown relative to control. D. ATP levels in PC3 (top) and ARCaP(M) (bottom) cells cultured in the absence or presence of CCM. Significant decrease in ATP levels was observed after 12 hours. CCM exposure had no effect on viability as shown by Calcein AM assay E. and JC-1 apoptosis analyses F. Values * P

    Techniques Used: Derivative Assay, Incubation, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Calcein AM Assay

    Atglistatin does not prevent lipid accumulation by the tumor cells and is not sufficient to prevent the induction of Warburg phenotype A. Immunofluorescence imaging of lipid droplets (BODIPY 493/503 nm) in PC3 cells alone (left panels) or in transwell co-culture with bone marrow adipocytes (right panels) and in the presence or absence of 10μM Atglistatin. DAPI was used as a nuclear stain; 40x images. Bar 100μm. B. Fluorescent intensity of the BODIPY 493/503 staining was quantified using Volocity (Perkin Elmer, Waltham, MA) and shown relative to PC3 cells alone. Results represent a mean of at least 3 independent experiments ± SD. C. Taqman RT-PCR analysis of ENO2, HK2 , and PDK1 in PC3 cells in transwell co-culture in the absence or presence of 10μM Atglistatin. Data are normalized to HPRT1 and shown relative to control. Values * P
    Figure Legend Snippet: Atglistatin does not prevent lipid accumulation by the tumor cells and is not sufficient to prevent the induction of Warburg phenotype A. Immunofluorescence imaging of lipid droplets (BODIPY 493/503 nm) in PC3 cells alone (left panels) or in transwell co-culture with bone marrow adipocytes (right panels) and in the presence or absence of 10μM Atglistatin. DAPI was used as a nuclear stain; 40x images. Bar 100μm. B. Fluorescent intensity of the BODIPY 493/503 staining was quantified using Volocity (Perkin Elmer, Waltham, MA) and shown relative to PC3 cells alone. Results represent a mean of at least 3 independent experiments ± SD. C. Taqman RT-PCR analysis of ENO2, HK2 , and PDK1 in PC3 cells in transwell co-culture in the absence or presence of 10μM Atglistatin. Data are normalized to HPRT1 and shown relative to control. Values * P

    Techniques Used: Immunofluorescence, Imaging, Co-Culture Assay, Staining, Reverse Transcription Polymerase Chain Reaction

    29) Product Images from "Characterization of the canine mda-7 gene, transcripts and expression patterns"

    Article Title: Characterization of the canine mda-7 gene, transcripts and expression patterns

    Journal: Gene

    doi: 10.1016/j.gene.2014.05.054

    Quantitative RT-PCR probe locations. TaqMan probes were designed against transcript specific sequences to detect sv2, sv3, sv4 and sv5. The copy number of canine mda- 7sv1 was deduced by subtracting the sum of the copy number of sv2, sv3, sv4 and sv5 from
    Figure Legend Snippet: Quantitative RT-PCR probe locations. TaqMan probes were designed against transcript specific sequences to detect sv2, sv3, sv4 and sv5. The copy number of canine mda- 7sv1 was deduced by subtracting the sum of the copy number of sv2, sv3, sv4 and sv5 from

    Techniques Used: Quantitative RT-PCR, Multiple Displacement Amplification

    30) Product Images from "Cidofovir selectivity is based on the different response of normal and cancer cells to DNA damage"

    Article Title: Cidofovir selectivity is based on the different response of normal and cancer cells to DNA damage

    Journal: BMC Medical Genomics

    doi: 10.1186/1755-8794-6-18

    Gene expression evaluated by real-time RT-PCR. ( A ) The TaqMan® Fast Universal PCR Master Mix and TaqMan® Gene Expression Assays from Applied Biosystems were employed to determine expression of the following genes (with assay ID’s): AOX1 (Hs00154079_m1), DHRS2 (Hs01061576_m1), HIST1H2AC (Hs00374312_s1), ICAM4 (Hs00169941_m1), MAP2K6 (Hs00992389_m1), OSMR (Hs00384276_m1), TP53 (Hs99999147_m1), and RB1 (Hs01078075_m1) in SiHa, HeLa, HaCaT, and PHKs following CDV treatment for 72 h. White bars represent the relative expression values of the microarray data. Black bars represent average RT-PCR values (±SD), relative to untreated cells and normalized against β–actin, from three independent samples. ( B ) Relative expression levels of HPV16 E6 and E7 in SiHa cells following treatment with CDV for different times, relative to untreated SiHa cells and normalized against β–actin. The bars show average values (±SD) of at least three independent experiments. An absolute 2-fold change difference was considered as biologically significant and is indicated by dashed lines in the figures.
    Figure Legend Snippet: Gene expression evaluated by real-time RT-PCR. ( A ) The TaqMan® Fast Universal PCR Master Mix and TaqMan® Gene Expression Assays from Applied Biosystems were employed to determine expression of the following genes (with assay ID’s): AOX1 (Hs00154079_m1), DHRS2 (Hs01061576_m1), HIST1H2AC (Hs00374312_s1), ICAM4 (Hs00169941_m1), MAP2K6 (Hs00992389_m1), OSMR (Hs00384276_m1), TP53 (Hs99999147_m1), and RB1 (Hs01078075_m1) in SiHa, HeLa, HaCaT, and PHKs following CDV treatment for 72 h. White bars represent the relative expression values of the microarray data. Black bars represent average RT-PCR values (±SD), relative to untreated cells and normalized against β–actin, from three independent samples. ( B ) Relative expression levels of HPV16 E6 and E7 in SiHa cells following treatment with CDV for different times, relative to untreated SiHa cells and normalized against β–actin. The bars show average values (±SD) of at least three independent experiments. An absolute 2-fold change difference was considered as biologically significant and is indicated by dashed lines in the figures.

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

    31) Product Images from "Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor"

    Article Title: Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34475-8

    Expression of GR in clinical lung adenocarcinoma and cell line models. A lung adenocarcinoma tissue microarray was probed by immunohistochemistry for expression of GR and the expression levels were scored in the malignant cells by intensity of staining as well as percent of cells that stained positive (Panel A). Representative images showing the different intensities of staining for GR are shown (Panel B). Whole cell lysates were extracted from H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells and GR expression was visualized by western blot using GAPDH as the loading control (Panel C). RNA was also extracted from the same cell lines and the levels of GR mRNA were measured by real time RT-PCR using TaqMan probes and plotted on a linear scale (Panel D). GR mRNA was measured by quantitative RT-PCR using SYBR Green in a cDNA microarray of clinical lung adenocarcinoma tumors; the relative levels of GR mRNA in H1299 and H1299GR Clone 4 cells were simultaneously measured using SYBR Green and the data plotted on a log scale (dashed lines) (Panel E). Panel D: *P, 0.002; **P, 0.001; ***P, 0.003.
    Figure Legend Snippet: Expression of GR in clinical lung adenocarcinoma and cell line models. A lung adenocarcinoma tissue microarray was probed by immunohistochemistry for expression of GR and the expression levels were scored in the malignant cells by intensity of staining as well as percent of cells that stained positive (Panel A). Representative images showing the different intensities of staining for GR are shown (Panel B). Whole cell lysates were extracted from H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells and GR expression was visualized by western blot using GAPDH as the loading control (Panel C). RNA was also extracted from the same cell lines and the levels of GR mRNA were measured by real time RT-PCR using TaqMan probes and plotted on a linear scale (Panel D). GR mRNA was measured by quantitative RT-PCR using SYBR Green in a cDNA microarray of clinical lung adenocarcinoma tumors; the relative levels of GR mRNA in H1299 and H1299GR Clone 4 cells were simultaneously measured using SYBR Green and the data plotted on a log scale (dashed lines) (Panel E). Panel D: *P, 0.002; **P, 0.001; ***P, 0.003.

    Techniques Used: Expressing, Microarray, Immunohistochemistry, Staining, Western Blot, Quantitative RT-PCR, SYBR Green Assay

    32) Product Images from "Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor"

    Article Title: Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34475-8

    Expression of GR in clinical lung adenocarcinoma and cell line models. A lung adenocarcinoma tissue microarray was probed by immunohistochemistry for expression of GR and the expression levels were scored in the malignant cells by intensity of staining as well as percent of cells that stained positive (Panel A). Representative images showing the different intensities of staining for GR are shown (Panel B). Whole cell lysates were extracted from H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells and GR expression was visualized by western blot using GAPDH as the loading control (Panel C). RNA was also extracted from the same cell lines and the levels of GR mRNA were measured by real time RT-PCR using TaqMan probes and plotted on a linear scale (Panel D). GR mRNA was measured by quantitative RT-PCR using SYBR Green in a cDNA microarray of clinical lung adenocarcinoma tumors; the relative levels of GR mRNA in H1299 and H1299GR Clone 4 cells were simultaneously measured using SYBR Green and the data plotted on a log scale (dashed lines) (Panel E). Panel D: *P, 0.002; **P, 0.001; ***P, 0.003.
    Figure Legend Snippet: Expression of GR in clinical lung adenocarcinoma and cell line models. A lung adenocarcinoma tissue microarray was probed by immunohistochemistry for expression of GR and the expression levels were scored in the malignant cells by intensity of staining as well as percent of cells that stained positive (Panel A). Representative images showing the different intensities of staining for GR are shown (Panel B). Whole cell lysates were extracted from H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells and GR expression was visualized by western blot using GAPDH as the loading control (Panel C). RNA was also extracted from the same cell lines and the levels of GR mRNA were measured by real time RT-PCR using TaqMan probes and plotted on a linear scale (Panel D). GR mRNA was measured by quantitative RT-PCR using SYBR Green in a cDNA microarray of clinical lung adenocarcinoma tumors; the relative levels of GR mRNA in H1299 and H1299GR Clone 4 cells were simultaneously measured using SYBR Green and the data plotted on a log scale (dashed lines) (Panel E). Panel D: *P, 0.002; **P, 0.001; ***P, 0.003.

    Techniques Used: Expressing, Microarray, Immunohistochemistry, Staining, Western Blot, Quantitative RT-PCR, SYBR Green Assay

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    Amplification:

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    Article Snippet: .. Total RNA was transcribed to cDNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Catalog # 4366597). cDNAs were amplified using the TaqMan® Fast Universal PCR Master Mix (2X), no AmpErase® UNG (Applied Biosystems, Catalog # 4352042). ..

    Real-time Polymerase Chain Reaction:

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    Article Title: Differential Roles of Peroxisome Proliferator-Activated Receptor-α and Receptor-γ on Renal Crystal Formation in Hyperoxaluric Rodents
    Article Snippet: .. Reactions were performed using TaqMan® Fast Universal PCR Master Mix (4352042; Applied Biosystems) and a 7500 Fast Real-Time PCR System (Applied Biosystems). .. The expression of each gene was normalized to that of a β -actin internal control (Actb ; Mm00607939_s1, or Rn00667869_m1).

    Polymerase Chain Reaction:

    Article Title: Increases in transient receptor potential vanilloid-1 mRNA and protein in primary afferent neurons stimulated by protein kinase C and their possible role in neurogenic inflammation
    Article Snippet: .. Real-time PCR was performed with Taqman reagents (TaqMan® Fast Universal PCR Master Mix, Applied Biosystems). .. All primers and probes with high efficiency were obtained from Applied Biosystems (Rn01460297 for rat TRPV1 , Rn00569199 for rat CGRP, and 4352931E for rat ACTB).

    Article Title: Extensive screening of microRNA populations identifies hsa-miR-375 and hsa-miR-133a-3p as selective markers for human rectal and colon cancer
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    Article Title: Bone marrow adipocytes promote tumor growth in bone via FABP4-dependent mechanisms
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    Article Title: Differential Roles of Peroxisome Proliferator-Activated Receptor-α and Receptor-γ on Renal Crystal Formation in Hyperoxaluric Rodents
    Article Snippet: .. Reactions were performed using TaqMan® Fast Universal PCR Master Mix (4352042; Applied Biosystems) and a 7500 Fast Real-Time PCR System (Applied Biosystems). .. The expression of each gene was normalized to that of a β -actin internal control (Actb ; Mm00607939_s1, or Rn00667869_m1).

    Article Title: Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity
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    Article Title: PPARγ Expression Is Diminished in Macrophages of Recurrent Miscarriage Placentas
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    Article Title: Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response
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    other:

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    Expressing:

    Article Title: Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity
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    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity
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    TaqMan Assay:

    Article Title: Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity
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    Thermo Fisher taqman fast universal pcr master mix
    30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) <t>Taqman</t> <t>PCR</t> assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p
    Taqman Fast Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman fast universal pcr master mix/product/Thermo Fisher
    Average 99 stars, based on 330 article reviews
    Price from $9.99 to $1999.99
    taqman fast universal pcr master mix - by Bioz Stars, 2020-07
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    91
    Thermo Fisher taqman fast universal pcr master mix 2×
    Probe and primer design for microfluidic quantitative <t>PCR</t> (MFQPCR) detection. Two assays (SET1 and SET2) were designed for each transgene. Forward and reverse primers for MFQPCR were designed to target different exons. <t>TaqMan</t> probe for MFQPCR was designed to target exon/exon junctions. Forward and reverse primers for pre-amplification were designed to include the forward and reverse primers for MFQPCR. While forward and reverse primers for pre-amplification and MFQPCR may amplify PCR products, including genomic DNA regions, TaqMan probes do not anneal to the PCR product, including genomic DNA regions ( A ). TaqMan probes specifically anneal to PCR products having exon/exon junction sequences ( B ).
    Taqman Fast Universal Pcr Master Mix 2×, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman fast universal pcr master mix 2×/product/Thermo Fisher
    Average 91 stars, based on 93 article reviews
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    taqman fast universal pcr master mix 2× - by Bioz Stars, 2020-07
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    Image Search Results


    30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) Taqman PCR assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p

    Journal: Aging (Albany NY)

    Article Title: Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13

    doi: 10.18632/aging.101270

    Figure Lengend Snippet: 30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) Taqman PCR assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p

    Article Snippet: Determination of miRNA expression of miR-125a-5p and miR-145b was done by using TaqMan micro RNA assay (ThermoFisher Scientific Catalog # 4427975), and TaqMan Fast Universal PCR master mix 2x (ThermoFisher Scientific Catalog # 4352042) as per the manufacturer's instructions.

    Techniques: Expressing, Microarray, Hybridization, Polymerase Chain Reaction, Mouse Assay

    Probe and primer design for microfluidic quantitative PCR (MFQPCR) detection. Two assays (SET1 and SET2) were designed for each transgene. Forward and reverse primers for MFQPCR were designed to target different exons. TaqMan probe for MFQPCR was designed to target exon/exon junctions. Forward and reverse primers for pre-amplification were designed to include the forward and reverse primers for MFQPCR. While forward and reverse primers for pre-amplification and MFQPCR may amplify PCR products, including genomic DNA regions, TaqMan probes do not anneal to the PCR product, including genomic DNA regions ( A ). TaqMan probes specifically anneal to PCR products having exon/exon junction sequences ( B ).

    Journal: Genes

    Article Title: Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control

    doi: 10.3390/genes11040457

    Figure Lengend Snippet: Probe and primer design for microfluidic quantitative PCR (MFQPCR) detection. Two assays (SET1 and SET2) were designed for each transgene. Forward and reverse primers for MFQPCR were designed to target different exons. TaqMan probe for MFQPCR was designed to target exon/exon junctions. Forward and reverse primers for pre-amplification were designed to include the forward and reverse primers for MFQPCR. While forward and reverse primers for pre-amplification and MFQPCR may amplify PCR products, including genomic DNA regions, TaqMan probes do not anneal to the PCR product, including genomic DNA regions ( A ). TaqMan probes specifically anneal to PCR products having exon/exon junction sequences ( B ).

    Article Snippet: For the sample mix, 1.8 μL of diluted pre-amplified PCR products were combined with 2.0 μL of TaqMan Fast Universal PCR Master Mix (2×), No AmpErase UNG (Thermo Fisher Scientific) and 0.2 μL of 20× GE Sample Loading Reagent (Fluidigm).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction