taqman universal pcr master mix kit  (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
    Catalog Number:
    4352042
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    Kits and Assays
    Applications:
    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 universal pcr master mix kit
    MuSK and agrin expression is increased in rat hippocampus after IA training. A , <t>TaqMan-PCR</t> analysis of MuSK on hippocampal extracts taken from rats trained in IA at 0 h−, 20 h−, and 20 h+. The concentration of mRNA was normalized against that of GAPDH. Data are expressed as mean fold change ± SEM of the ratio 20 h+:0 h− versus 20 h−:0 h− (∗ 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 universal pcr master mix kit/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    taqman universal pcr master mix kit - by Bioz Stars, 2021-07
    99/100 stars

    Images

    1) Product Images from "MuSK Expressed in the Brain Mediates Cholinergic Responses, Synaptic Plasticity, and Memory Formation"

    Article Title: MuSK Expressed in the Brain Mediates Cholinergic Responses, Synaptic Plasticity, and Memory Formation

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1674-06.2006

    MuSK and agrin expression is increased in rat hippocampus after IA training. A , TaqMan-PCR analysis of MuSK on hippocampal extracts taken from rats trained in IA at 0 h−, 20 h−, and 20 h+. The concentration of mRNA was normalized against that of GAPDH. Data are expressed as mean fold change ± SEM of the ratio 20 h+:0 h− versus 20 h−:0 h− (∗ p
    Figure Legend Snippet: MuSK and agrin expression is increased in rat hippocampus after IA training. A , TaqMan-PCR analysis of MuSK on hippocampal extracts taken from rats trained in IA at 0 h−, 20 h−, and 20 h+. The concentration of mRNA was normalized against that of GAPDH. Data are expressed as mean fold change ± SEM of the ratio 20 h+:0 h− versus 20 h−:0 h− (∗ p

    Techniques Used: Expressing, IA, Polymerase Chain Reaction, Concentration Assay

    2) Product Images from "B-vitamin deficiency is protective against DSS-induced colitis in mice"

    Article Title: B-vitamin deficiency is protective against DSS-induced colitis in mice

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00076.2011

    Gene expression changes. Mice were given the control or B 6 -B 12 -deficient diet for 2 wk followed by 5 days of 3% DSS. After euthanasia, colonic RNA was extracted for gene expression changes using TaqMan probes for real-time PCR (Con n = 10, Def n = 10, Con+3% n = 9, Def+3% n = 8). As expected, expression of TNF-α, inducible nitric oxide synthase (iNOS), and IL-10 were significantly increased in DSS mice compared with non-DSS controls ( P
    Figure Legend Snippet: Gene expression changes. Mice were given the control or B 6 -B 12 -deficient diet for 2 wk followed by 5 days of 3% DSS. After euthanasia, colonic RNA was extracted for gene expression changes using TaqMan probes for real-time PCR (Con n = 10, Def n = 10, Con+3% n = 9, Def+3% n = 8). As expected, expression of TNF-α, inducible nitric oxide synthase (iNOS), and IL-10 were significantly increased in DSS mice compared with non-DSS controls ( P

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

    3) Product Images from "The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability"

    Article Title: The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability

    Journal: Biochimica et Biophysica Acta

    doi: 10.1016/j.bbamcr.2017.09.009

    CKS1B and CKS2 expression is higher in diagnostic bone marrow samples from patients carrying MLL -translocations compared to PBMCs from healthy controls. RNA extracted from human umbilical cord blood CD34 + cells, PBMCs from healthy donors, and leukaemic patient samples, were assessed for (A) CKS1B and (B) CKS2 expression by TaqMan quantitative PCR. Three independent control genes were used ( ABL , GUS , B2M ) to measure relative RNA abundance. Values are represented as relative gene expression versus GUS control. Each point represents one patient. Median bars with standard deviation are presented for each set of samples. A Student's t -test was used to assess significance, and * indicates significance versus CD34 + cells, and § indicates significance versus PBMCs.
    Figure Legend Snippet: CKS1B and CKS2 expression is higher in diagnostic bone marrow samples from patients carrying MLL -translocations compared to PBMCs from healthy controls. RNA extracted from human umbilical cord blood CD34 + cells, PBMCs from healthy donors, and leukaemic patient samples, were assessed for (A) CKS1B and (B) CKS2 expression by TaqMan quantitative PCR. Three independent control genes were used ( ABL , GUS , B2M ) to measure relative RNA abundance. Values are represented as relative gene expression versus GUS control. Each point represents one patient. Median bars with standard deviation are presented for each set of samples. A Student's t -test was used to assess significance, and * indicates significance versus CD34 + cells, and § indicates significance versus PBMCs.

    Techniques Used: Expressing, Diagnostic Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    4) Product Images from "Preventative oral methylthioadenosine is anti-inflammatory and reduces DSS-induced colitis in mice"

    Article Title: Preventative oral methylthioadenosine is anti-inflammatory and reduces DSS-induced colitis in mice

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00549.2011

    MTA reduces upregulation of gene expression. Mice were given 3% DSS for 5 days and 150 mg/kg body wt MTA for 7 days via the drinking water. After euthanasia, colonic RNA was extracted for gene expression changes using TaqMan probes for real-time PCR (Con
    Figure Legend Snippet: MTA reduces upregulation of gene expression. Mice were given 3% DSS for 5 days and 150 mg/kg body wt MTA for 7 days via the drinking water. After euthanasia, colonic RNA was extracted for gene expression changes using TaqMan probes for real-time PCR (Con

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

    Related Articles

    other:

    Article Title: MicroRNA Expression Changes during Interferon-Beta Treatment in the Peripheral Blood of Multiple Sclerosis Patients
    Article Snippet: The validation experiment was performed using TaqMan single-tube assays for hsa-miR-16-5p (Assay ID 000391), hsa-miR-29a-3p (Assay ID 002112), hsa-miR-29c-3p (Assay ID 000587), hsa-miR-149-5p (Assay ID 002255), and hsa-miR-532-5p (Assay ID 001518).

    Amplification:

    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
    Article Snippet: MiRNA expression was assessed using the TaqMan® MicroRNA Assays from Applied Biosystems following the manufacturer’s instructions. .. 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). ..

    Polymerase Chain Reaction:

    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
    Article Snippet: MiRNA expression was assessed using the TaqMan® MicroRNA Assays from Applied Biosystems following the manufacturer’s instructions. .. 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). ..

    Article Title: MuSK Expressed in the Brain Mediates Cholinergic Responses, Synaptic Plasticity, and Memory Formation
    Article Snippet: Hippocampal total RNA was extracted with TRIzol (Invitrogen) and reverse transcribed into cDNAs using Omniscript reverse transcriptase (Qiagen, Hilden, Germany). .. TaqMan 5′ nuclease fluorogenic quantitative PCR assays were performed using the TaqMan Universal PCR Master Mix kit (PE Applied Biosystems, Foster City, CA) according to manufacturer’s instructions. .. Probes and primers were designed using PrimerExpress3 software (PE Applied Biosystems).

    Article Title: Unusual Five Copies and Dual Forms of nrdB in “Candidatus Liberibacter asiaticus”: Biological Implications and PCR Detection Application
    Article Snippet: The fluorescence signal was captured at the end of each 60 °C step, followed by a melting point analysis. .. For TaqMan® real-time PCR, the reaction mixture contained 10 μl of TaqMan® Fast Universal PCR Master Mix (2X) (Applied Biosystems), 1 μl of DNA template (~25 ng), 0.2 μl of TaqMan® probe (5 μM), 0.4 μl of each forward and reverse primer (10 μM) in a final volume of 20 μl with the following procedure: 50 °C for 2 min, then 95 °C for 20 s, followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. The fluorescence signal was captured at the end of each 60 °C step. .. The data were analyzed using Opticon Monitor™ software (MJ Research), StepOnePlus™ Software v2.3 (Applied Biosystems) and Bio-Rad CFX Manager 2.1 software with automated baseline settings and a manually set threshold at 0.1.

    Article Title: Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13
    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. .. The detection system used was CFX96 qPCR (BioRad).

    Article Title: Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation
    Article Snippet: For the detection of M ϕ -related genes, we used probe sets for Emr1 (Mm00802529_m1), Cd68 (Mm03047340_m1), Itgax (Mm00498698_m1), Nos2 (Mm00440502_m1), Tnf (Mm00443258_m1), Il 6 (Mm00446190_m1), Cd 163 (Mm00474091_m1), Mrc1 (Mm00485148_m1), Arg1 (Mm00475988_m1), Chi3l3 (Mm04213363_u1), and Il 10 (Mm00439614_m1). .. Quantitative PCR was performed using a TaqMan FAST Universal PCR Master Mix (4352042; Applied Biosystems) with a 7500 FAST Real-Time PCR System (Applied Biosystems). .. The expression of each gene was normalized to the expression of β -actin, which was used as the internal control.

    Real-time Polymerase Chain Reaction:

    Article Title: MuSK Expressed in the Brain Mediates Cholinergic Responses, Synaptic Plasticity, and Memory Formation
    Article Snippet: Hippocampal total RNA was extracted with TRIzol (Invitrogen) and reverse transcribed into cDNAs using Omniscript reverse transcriptase (Qiagen, Hilden, Germany). .. TaqMan 5′ nuclease fluorogenic quantitative PCR assays were performed using the TaqMan Universal PCR Master Mix kit (PE Applied Biosystems, Foster City, CA) according to manufacturer’s instructions. .. Probes and primers were designed using PrimerExpress3 software (PE Applied Biosystems).

    Article Title: Robust Self-Renewal of Rat Embryonic Stem Cells Requires Fine-Tuning of Glycogen Synthase Kinase-3 Inhibition
    Article Snippet: For rat embryos, a pool of 14 embryonic day 5.5 rat blastocysts was harvested and homogenized using QIAshredder (QIAGEN) prior to total RNA extraction. .. For real-time PCR, we used TaqMan Fast Universal Master Mix and TaqMan probes (Applied Biosystems) or Fast SYBR Green Master Mix and primers ( ). .. RNA Interference Rat ESCs were transfected with siRNA at a final concentration of 40 nM using Dharmafect 1 (Dharmacon, cat. T-2001-01) and then replated at a concentration of 30,000 cells per well in 12-well plates.

    Article Title: Unusual Five Copies and Dual Forms of nrdB in “Candidatus Liberibacter asiaticus”: Biological Implications and PCR Detection Application
    Article Snippet: The fluorescence signal was captured at the end of each 60 °C step, followed by a melting point analysis. .. For TaqMan® real-time PCR, the reaction mixture contained 10 μl of TaqMan® Fast Universal PCR Master Mix (2X) (Applied Biosystems), 1 μl of DNA template (~25 ng), 0.2 μl of TaqMan® probe (5 μM), 0.4 μl of each forward and reverse primer (10 μM) in a final volume of 20 μl with the following procedure: 50 °C for 2 min, then 95 °C for 20 s, followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. The fluorescence signal was captured at the end of each 60 °C step. .. The data were analyzed using Opticon Monitor™ software (MJ Research), StepOnePlus™ Software v2.3 (Applied Biosystems) and Bio-Rad CFX Manager 2.1 software with automated baseline settings and a manually set threshold at 0.1.

    Article Title: Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation
    Article Snippet: For the detection of M ϕ -related genes, we used probe sets for Emr1 (Mm00802529_m1), Cd68 (Mm03047340_m1), Itgax (Mm00498698_m1), Nos2 (Mm00440502_m1), Tnf (Mm00443258_m1), Il 6 (Mm00446190_m1), Cd 163 (Mm00474091_m1), Mrc1 (Mm00485148_m1), Arg1 (Mm00475988_m1), Chi3l3 (Mm04213363_u1), and Il 10 (Mm00439614_m1). .. Quantitative PCR was performed using a TaqMan FAST Universal PCR Master Mix (4352042; Applied Biosystems) with a 7500 FAST Real-Time PCR System (Applied Biosystems). .. The expression of each gene was normalized to the expression of β -actin, which was used as the internal control.

    SYBR Green Assay:

    Article Title: Robust Self-Renewal of Rat Embryonic Stem Cells Requires Fine-Tuning of Glycogen Synthase Kinase-3 Inhibition
    Article Snippet: For rat embryos, a pool of 14 embryonic day 5.5 rat blastocysts was harvested and homogenized using QIAshredder (QIAGEN) prior to total RNA extraction. .. For real-time PCR, we used TaqMan Fast Universal Master Mix and TaqMan probes (Applied Biosystems) or Fast SYBR Green Master Mix and primers ( ). .. RNA Interference Rat ESCs were transfected with siRNA at a final concentration of 40 nM using Dharmafect 1 (Dharmacon, cat. T-2001-01) and then replated at a concentration of 30,000 cells per well in 12-well plates.

    Fluorescence:

    Article Title: Unusual Five Copies and Dual Forms of nrdB in “Candidatus Liberibacter asiaticus”: Biological Implications and PCR Detection Application
    Article Snippet: The fluorescence signal was captured at the end of each 60 °C step, followed by a melting point analysis. .. For TaqMan® real-time PCR, the reaction mixture contained 10 μl of TaqMan® Fast Universal PCR Master Mix (2X) (Applied Biosystems), 1 μl of DNA template (~25 ng), 0.2 μl of TaqMan® probe (5 μM), 0.4 μl of each forward and reverse primer (10 μM) in a final volume of 20 μl with the following procedure: 50 °C for 2 min, then 95 °C for 20 s, followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. The fluorescence signal was captured at the end of each 60 °C step. .. The data were analyzed using Opticon Monitor™ software (MJ Research), StepOnePlus™ Software v2.3 (Applied Biosystems) and Bio-Rad CFX Manager 2.1 software with automated baseline settings and a manually set threshold at 0.1.

    Expressing:

    Article Title: Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13
    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. .. The detection system used was CFX96 qPCR (BioRad).

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    Thermo Fisher taqman universal pcr master mix
    MiRNA validation. Nineteen miRNAs from our deep sequencing data were selected for further validation using real-time <t>TaqMan®</t> MicroRNA Assays, including nine novel, cat-specific miRNAs. miR-151 and miR-361 were expressed at very consistent levels in all of the deep sequencing samples (not shown) and, thus, <t>qRT-PCR</t> values were normalized to these two miRs. Figure shows heat maps of averaged and normalized miR counts from the deep sequencing data and of the normalized relative expression from qRT-PCR. All of the normalized deep sequencing and qRT-PCR values for each individual miR in each individual sample are given in Supplementary Table S1.10 .
    Taqman Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman universal pcr master mix/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    taqman universal pcr master mix - by Bioz Stars, 2021-07
    99/100 stars
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    99
    Thermo Fisher taqman universal master mix ii
    Expression of CIC and its targets in human GBM tumors and cells. Human operative GBM samples or normal derived brains (NB) were lysed and a immunoblotted with indicated antibodies b or total RNA extracted and quantitative real-time <t>PCR</t> analysis was carried out using <t>TaqMan</t> gene expression assays. The graph depicts fold changes in CIC expression relative to normal brain. c Nuclear or cytoplasmic lysates were isolated from human operative GBM samples or normal brain and were immunoblotted with indicated antibodies. d Human operative astrocytoma samples were lysed and immunoblotted with indicated antibodies. Human-derived GBM cell lines or normal human astrocytes (NHA) were lysed and e protein lysates were immunoblotted with indicated antibodies ( f ) or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to NHA. Data represent mean ± s.e.m. of three independent experiments performed in triplicate. * P
    Taqman Universal Master Mix Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman universal master mix ii/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    MiRNA validation. Nineteen miRNAs from our deep sequencing data were selected for further validation using real-time TaqMan® MicroRNA Assays, including nine novel, cat-specific miRNAs. miR-151 and miR-361 were expressed at very consistent levels in all of the deep sequencing samples (not shown) and, thus, qRT-PCR values were normalized to these two miRs. Figure shows heat maps of averaged and normalized miR counts from the deep sequencing data and of the normalized relative expression from qRT-PCR. All of the normalized deep sequencing and qRT-PCR values for each individual miR in each individual sample are given in Supplementary Table S1.10 .

    Journal: Scientific Reports

    Article Title: Discovery and characterization of the feline miRNAome

    doi: 10.1038/s41598-017-10164-w

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

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

    Techniques: Sequencing, Quantitative RT-PCR, Expressing

    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.

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-34475-8

    Figure Lengend 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.

    Article Snippet: Reverse transcription was performed using 500 ng of total RNA and High-Capacity cDNA Archive kit (Thermo Fisher Scientific), according to the vendor’s protocol. cDNA was measured by quantitative real-time PCR using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) and TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific).

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky273

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

    Article Snippet: The TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) was used to quantify mRNA expression of MDA5, IFIT1, IFIT5 and GAPDH with TaqMan Gene Expression Assays specific to these genes.

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

    Expression of CIC and its targets in human GBM tumors and cells. Human operative GBM samples or normal derived brains (NB) were lysed and a immunoblotted with indicated antibodies b or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to normal brain. c Nuclear or cytoplasmic lysates were isolated from human operative GBM samples or normal brain and were immunoblotted with indicated antibodies. d Human operative astrocytoma samples were lysed and immunoblotted with indicated antibodies. Human-derived GBM cell lines or normal human astrocytes (NHA) were lysed and e protein lysates were immunoblotted with indicated antibodies ( f ) or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to NHA. Data represent mean ± s.e.m. of three independent experiments performed in triplicate. * P

    Journal: Nature Communications

    Article Title: CIC protein instability contributes to tumorigenesis in glioblastoma

    doi: 10.1038/s41467-018-08087-9

    Figure Lengend Snippet: Expression of CIC and its targets in human GBM tumors and cells. Human operative GBM samples or normal derived brains (NB) were lysed and a immunoblotted with indicated antibodies b or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to normal brain. c Nuclear or cytoplasmic lysates were isolated from human operative GBM samples or normal brain and were immunoblotted with indicated antibodies. d Human operative astrocytoma samples were lysed and immunoblotted with indicated antibodies. Human-derived GBM cell lines or normal human astrocytes (NHA) were lysed and e protein lysates were immunoblotted with indicated antibodies ( f ) or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to NHA. Data represent mean ± s.e.m. of three independent experiments performed in triplicate. * P

    Article Snippet: One μg RNA was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) and quantitative real-time PCR performed was performed using TaqMan universal master mix II (Life Technologies) according the manufacturers protocol with a StepOne Real-Time PCR machine (Life Technologies).

    Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Isolation