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  • 99
    Thermo Fisher trizol reagent trizol
    Cellular assembly of hTRmin telomerase. ( A ) U2OS cells were transiently transfected with constructs encoding F-TERT and either hTRmin, hTR, or empty vector (mock). TERT was immunopurified on anti-FLAG agarose from cell extract 48 hr post-transfection. An aliquot of the bound samples were treated with <t>TRIzol</t> to purify <t>RNA,</t> which was analyzed by Northern blot. In parallel, bound samples were tested for telomerase activity by primer extension using 32 P dGTP for radiolabeling. Products were precipitated and resolved by denaturing gel electrophoresis. A radioactive oligonucleotide recovery control (RC) was added prior to precipitation. For the hTR cell extract, different amounts of extract were assayed relative to the hTRmin sample set at 100%. All lanes are from the same gel. ( B ) HCT116 hTR KO#2 cells were transiently transfected to express hTR, CAB-box-mutant hTR (G414C), LhTRmin, or hTRmin. FISH was performed to detect the RNAs (green). Nuclei were counterstained with DAPI (blue). DOI: http://dx.doi.org/10.7554/eLife.18221.004
    Trizol Reagent Trizol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 64517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher rna trizol
    Cellular assembly of hTRmin telomerase. ( A ) U2OS cells were transiently transfected with constructs encoding F-TERT and either hTRmin, hTR, or empty vector (mock). TERT was immunopurified on anti-FLAG agarose from cell extract 48 hr post-transfection. An aliquot of the bound samples were treated with <t>TRIzol</t> to purify <t>RNA,</t> which was analyzed by Northern blot. In parallel, bound samples were tested for telomerase activity by primer extension using 32 P dGTP for radiolabeling. Products were precipitated and resolved by denaturing gel electrophoresis. A radioactive oligonucleotide recovery control (RC) was added prior to precipitation. For the hTR cell extract, different amounts of extract were assayed relative to the hTRmin sample set at 100%. All lanes are from the same gel. ( B ) HCT116 hTR KO#2 cells were transiently transfected to express hTR, CAB-box-mutant hTR (G414C), LhTRmin, or hTRmin. FISH was performed to detect the RNAs (green). Nuclei were counterstained with DAPI (blue). DOI: http://dx.doi.org/10.7554/eLife.18221.004
    Rna Trizol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher trizol lysis
    Cellular assembly of hTRmin telomerase. ( A ) U2OS cells were transiently transfected with constructs encoding F-TERT and either hTRmin, hTR, or empty vector (mock). TERT was immunopurified on anti-FLAG agarose from cell extract 48 hr post-transfection. An aliquot of the bound samples were treated with <t>TRIzol</t> to purify <t>RNA,</t> which was analyzed by Northern blot. In parallel, bound samples were tested for telomerase activity by primer extension using 32 P dGTP for radiolabeling. Products were precipitated and resolved by denaturing gel electrophoresis. A radioactive oligonucleotide recovery control (RC) was added prior to precipitation. For the hTR cell extract, different amounts of extract were assayed relative to the hTRmin sample set at 100%. All lanes are from the same gel. ( B ) HCT116 hTR KO#2 cells were transiently transfected to express hTR, CAB-box-mutant hTR (G414C), LhTRmin, or hTRmin. FISH was performed to detect the RNAs (green). Nuclei were counterstained with DAPI (blue). DOI: http://dx.doi.org/10.7554/eLife.18221.004
    Trizol Lysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ls trizol
    Cellular assembly of hTRmin telomerase. ( A ) U2OS cells were transiently transfected with constructs encoding F-TERT and either hTRmin, hTR, or empty vector (mock). TERT was immunopurified on anti-FLAG agarose from cell extract 48 hr post-transfection. An aliquot of the bound samples were treated with <t>TRIzol</t> to purify <t>RNA,</t> which was analyzed by Northern blot. In parallel, bound samples were tested for telomerase activity by primer extension using 32 P dGTP for radiolabeling. Products were precipitated and resolved by denaturing gel electrophoresis. A radioactive oligonucleotide recovery control (RC) was added prior to precipitation. For the hTR cell extract, different amounts of extract were assayed relative to the hTRmin sample set at 100%. All lanes are from the same gel. ( B ) HCT116 hTR KO#2 cells were transiently transfected to express hTR, CAB-box-mutant hTR (G414C), LhTRmin, or hTRmin. FISH was performed to detect the RNAs (green). Nuclei were counterstained with DAPI (blue). DOI: http://dx.doi.org/10.7554/eLife.18221.004
    Ls Trizol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cellular assembly of hTRmin telomerase. ( A ) U2OS cells were transiently transfected with constructs encoding F-TERT and either hTRmin, hTR, or empty vector (mock). TERT was immunopurified on anti-FLAG agarose from cell extract 48 hr post-transfection. An aliquot of the bound samples were treated with TRIzol to purify RNA, which was analyzed by Northern blot. In parallel, bound samples were tested for telomerase activity by primer extension using 32 P dGTP for radiolabeling. Products were precipitated and resolved by denaturing gel electrophoresis. A radioactive oligonucleotide recovery control (RC) was added prior to precipitation. For the hTR cell extract, different amounts of extract were assayed relative to the hTRmin sample set at 100%. All lanes are from the same gel. ( B ) HCT116 hTR KO#2 cells were transiently transfected to express hTR, CAB-box-mutant hTR (G414C), LhTRmin, or hTRmin. FISH was performed to detect the RNAs (green). Nuclei were counterstained with DAPI (blue). DOI: http://dx.doi.org/10.7554/eLife.18221.004

    Journal: eLife

    Article Title: Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies

    doi: 10.7554/eLife.18221

    Figure Lengend Snippet: Cellular assembly of hTRmin telomerase. ( A ) U2OS cells were transiently transfected with constructs encoding F-TERT and either hTRmin, hTR, or empty vector (mock). TERT was immunopurified on anti-FLAG agarose from cell extract 48 hr post-transfection. An aliquot of the bound samples were treated with TRIzol to purify RNA, which was analyzed by Northern blot. In parallel, bound samples were tested for telomerase activity by primer extension using 32 P dGTP for radiolabeling. Products were precipitated and resolved by denaturing gel electrophoresis. A radioactive oligonucleotide recovery control (RC) was added prior to precipitation. For the hTR cell extract, different amounts of extract were assayed relative to the hTRmin sample set at 100%. All lanes are from the same gel. ( B ) HCT116 hTR KO#2 cells were transiently transfected to express hTR, CAB-box-mutant hTR (G414C), LhTRmin, or hTRmin. FISH was performed to detect the RNAs (green). Nuclei were counterstained with DAPI (blue). DOI: http://dx.doi.org/10.7554/eLife.18221.004

    Article Snippet: Northern blots RNA was purified using TRIzol (Thermo) and resuspended in distilled water.

    Techniques: Transfection, Construct, Plasmid Preparation, Northern Blot, Activity Assay, Radioactivity, Nucleic Acid Electrophoresis, Mutagenesis, Fluorescence In Situ Hybridization

    Cytokine gene expression is reduced during CHIKV Opal524R infection. (A, B) Twenty-four-day-old C57BL6/J mice were infected in the left rear footpad with 100 PFU of wild-type CHIKV ( n = 5) or Opal524R ( n = 5) or mock infected with diluent ( n = 3). At days 1 (A) and 2 (B) postinfection, mice were sacrificed and perfused intracardially with 1× PBS, and the ipsilateral foot was harvested in TRIzol. RNA was extracted, cDNA was generated, and cytokine gene expression was assessed by quantitative real-time PCR. Each cytokine was normalized to host 18S gene expression. Fold changes in gene expression relative to mock infection (left) and wild-type infection (right) are shown. Data are representative of two independent experiments. Statistical significance is indicated as follows: *, P

    Journal: mBio

    Article Title: Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology

    doi: 10.1128/mBio.01456-17

    Figure Lengend Snippet: Cytokine gene expression is reduced during CHIKV Opal524R infection. (A, B) Twenty-four-day-old C57BL6/J mice were infected in the left rear footpad with 100 PFU of wild-type CHIKV ( n = 5) or Opal524R ( n = 5) or mock infected with diluent ( n = 3). At days 1 (A) and 2 (B) postinfection, mice were sacrificed and perfused intracardially with 1× PBS, and the ipsilateral foot was harvested in TRIzol. RNA was extracted, cDNA was generated, and cytokine gene expression was assessed by quantitative real-time PCR. Each cytokine was normalized to host 18S gene expression. Fold changes in gene expression relative to mock infection (left) and wild-type infection (right) are shown. Data are representative of two independent experiments. Statistical significance is indicated as follows: *, P

    Article Snippet: RNA was isolated from purified virus preparations by TRIzol extraction (Ambion).

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

    Recovery of nucleic acids and polyphosphate (polyP) using various purification methods. Known quantities of bacteriophage lambda DNA (DNA, striped bars), polyG (RNA, white bars), or long-chain polyP (black bars) were processed for nucleic acid purification using Qiaquick, DNeasy, or TRIzol kits, following the manufacturer’s instructions, or with phenol/chloroform extraction. Nucleic acid recovery was quantified using A 260 and polyP recovery by digestion with PPX1 and PiBlue assay. Results are plotted as percent of the input amount in each experiment. For all experiments except the Qiaquick kits, the three samples were: (A) DNA alone (5 µg lambda DNA); (B) RNA alone (5 µg polyG); (C) 5 µg polyP alone; (D) a mixture of 5 µg polyP plus 5 µg of DNA; or (E) 5 µg polyP plus 5 µg of RNA (E) . Experiments with the Qiaquick kit employed 2.5 µg of polyP and/or nucleic acid. Data are mean ± SEM ( n = 3).

    Journal: Frontiers in Medicine

    Article Title: Ability of Polyphosphate and Nucleic Acids to Trigger Blood Clotting: Some Observations and Caveats

    doi: 10.3389/fmed.2018.00107

    Figure Lengend Snippet: Recovery of nucleic acids and polyphosphate (polyP) using various purification methods. Known quantities of bacteriophage lambda DNA (DNA, striped bars), polyG (RNA, white bars), or long-chain polyP (black bars) were processed for nucleic acid purification using Qiaquick, DNeasy, or TRIzol kits, following the manufacturer’s instructions, or with phenol/chloroform extraction. Nucleic acid recovery was quantified using A 260 and polyP recovery by digestion with PPX1 and PiBlue assay. Results are plotted as percent of the input amount in each experiment. For all experiments except the Qiaquick kits, the three samples were: (A) DNA alone (5 µg lambda DNA); (B) RNA alone (5 µg polyG); (C) 5 µg polyP alone; (D) a mixture of 5 µg polyP plus 5 µg of DNA; or (E) 5 µg polyP plus 5 µg of RNA (E) . Experiments with the Qiaquick kit employed 2.5 µg of polyP and/or nucleic acid. Data are mean ± SEM ( n = 3).

    Article Snippet: In these experiments, known quantities of purified bacteriophage lambda DNA or a synthetic RNA homopolymer, polyG, either alone or mixed with long-chain polyP, were purified using phenol/chloroform extraction or Qiaquick kits (Qiagen), DNeasy kits (Qiagen), RNeasy Miniprep kits (Qiagen), or TRIzol kits (Thermo Fisher), following the manufacturers’ instructions.

    Techniques: Purification, Lambda DNA Preparation, Nucleic Acid Purification

    Simultaneous optimization of EDTA, NaCl, and SDS concentrations enhances protein yield during solubilization of TRIzol-precipitated protein

    Journal: Journal of neuroscience methods

    Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

    doi: 10.1016/j.jneumeth.2017.02.002

    Figure Lengend Snippet: Simultaneous optimization of EDTA, NaCl, and SDS concentrations enhances protein yield during solubilization of TRIzol-precipitated protein

    Article Snippet: In fact, the TRIzol protocol (Life Technologies) recommends solubilization in 1% SDS, and indicates that dialysis and subsequent reconstitution in an SDS-urea solution may improve yield (Life Technologies; November 9, 2016 edition).

    Techniques:

    3.1 EDTA, NaCl, and SDS concentrations modulate protein yield during solubilization of TRIzol-precipitated protein

    Journal: Journal of neuroscience methods

    Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

    doi: 10.1016/j.jneumeth.2017.02.002

    Figure Lengend Snippet: 3.1 EDTA, NaCl, and SDS concentrations modulate protein yield during solubilization of TRIzol-precipitated protein

    Article Snippet: In fact, the TRIzol protocol (Life Technologies) recommends solubilization in 1% SDS, and indicates that dialysis and subsequent reconstitution in an SDS-urea solution may improve yield (Life Technologies; November 9, 2016 edition).

    Techniques:

    EDTA, NaCl, and SDS concentrations significantly alter protein yield during solubilization of TRIzol-precipitated protein

    Journal: Journal of neuroscience methods

    Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

    doi: 10.1016/j.jneumeth.2017.02.002

    Figure Lengend Snippet: EDTA, NaCl, and SDS concentrations significantly alter protein yield during solubilization of TRIzol-precipitated protein

    Article Snippet: In fact, the TRIzol protocol (Life Technologies) recommends solubilization in 1% SDS, and indicates that dialysis and subsequent reconstitution in an SDS-urea solution may improve yield (Life Technologies; November 9, 2016 edition).

    Techniques:

    Expression of miR-15a and miR-16 and correlation with Bmi-1 levels in ovarian cancer cell lines and patient samples. (A) Total RNA was collected from the ovarian cancer cell lines using the TRIzol method and subjected to RT-PCR. The comparative C t method

    Journal:

    Article Title: MiR-15a and MiR-16 control Bmi-1 expression in ovarian cancer

    doi: 10.1158/0008-5472.CAN-09-2552

    Figure Lengend Snippet: Expression of miR-15a and miR-16 and correlation with Bmi-1 levels in ovarian cancer cell lines and patient samples. (A) Total RNA was collected from the ovarian cancer cell lines using the TRIzol method and subjected to RT-PCR. The comparative C t method

    Article Snippet: Total RNA was isolated from transfected cells using TRIzol reagent (Invitrogen).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    THSG inhibited vascular-remodelling-related gene expression. Total RNA was extracted from rat aortas by using Invitrogen TRIzol reagent. ((a) to (l)) COL1A2, ACTCA, TIMP1, CCL3, IGFBP1, ECE1, KLF5, MYL1, WISP2, COL3A1, BMP4, and FN1 mRNA expression was measured using a Maxima SYBR Green qPCR Master Mix kit. Data are normalised to GAPDH mRNA expression levels ( n = 4). Data are expressed as the mean ± SEM. ∗ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Aortic Remodelling Is Improved by 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside Involving the Smad3 Pathway in Spontaneously Hypertensive Rats

    doi: 10.1155/2015/789027

    Figure Lengend Snippet: THSG inhibited vascular-remodelling-related gene expression. Total RNA was extracted from rat aortas by using Invitrogen TRIzol reagent. ((a) to (l)) COL1A2, ACTCA, TIMP1, CCL3, IGFBP1, ECE1, KLF5, MYL1, WISP2, COL3A1, BMP4, and FN1 mRNA expression was measured using a Maxima SYBR Green qPCR Master Mix kit. Data are normalised to GAPDH mRNA expression levels ( n = 4). Data are expressed as the mean ± SEM. ∗ P

    Article Snippet: RNA Isolation and Quantitative PCR Total RNA was extracted from the rat aortas with the Invitrogen TRIzol reagent (Life Technologies, Grand Island, USA) according to the manufacturer's instructions.

    Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction

    NR1D2 is directly targeted by miR-210. ( A ) Putative binding sites for human miR-210 were predicted in the 3′-UTR of NR1D2 mRNA. The mutated bases of predicted miR-210 binding sites are underlined, namely MT-NR1D2 3′-UTR. ( B ) miR-210 targeted the 3′-UTR of NR1D2. Luciferase reporters containing either miR-210 putative binding sites from the wild-type NR1D2 3′-UTR (WT NR1D2-3′-UTR) or mutated NR1D2 3′-UTR (MT NR1D2-3′-UTR) were co-transfected with the indicated microRNA mimics into 293T cells. Luciferase activity was measured 30 h after transfection. ( C ) The NR1D2 protein was quantified by Western blotting. NT-2 cells were harvested 48 h after transfection with RIPA lysis buffer. Gels were run under the same experimental conditions (120 V for 90 mins). ( D ) NR1D2 mRNA expression was evaluated by real-time PCR. Total RNA was extracted 48 h after transfection using a standard TRIzol protocol. WT: wild-type, MT: mutant, MNC: normal control mimics, M210: miR-210 mimics, INC: normal control inhibitors, I210: miR-210 inhibitors. All data are presented as the means ± SEMs from at least three independent experiments. *p

    Journal: Scientific Reports

    Article Title: The functional and predictive roles of miR-210 in cryptorchidism

    doi: 10.1038/srep32265

    Figure Lengend Snippet: NR1D2 is directly targeted by miR-210. ( A ) Putative binding sites for human miR-210 were predicted in the 3′-UTR of NR1D2 mRNA. The mutated bases of predicted miR-210 binding sites are underlined, namely MT-NR1D2 3′-UTR. ( B ) miR-210 targeted the 3′-UTR of NR1D2. Luciferase reporters containing either miR-210 putative binding sites from the wild-type NR1D2 3′-UTR (WT NR1D2-3′-UTR) or mutated NR1D2 3′-UTR (MT NR1D2-3′-UTR) were co-transfected with the indicated microRNA mimics into 293T cells. Luciferase activity was measured 30 h after transfection. ( C ) The NR1D2 protein was quantified by Western blotting. NT-2 cells were harvested 48 h after transfection with RIPA lysis buffer. Gels were run under the same experimental conditions (120 V for 90 mins). ( D ) NR1D2 mRNA expression was evaluated by real-time PCR. Total RNA was extracted 48 h after transfection using a standard TRIzol protocol. WT: wild-type, MT: mutant, MNC: normal control mimics, M210: miR-210 mimics, INC: normal control inhibitors, I210: miR-210 inhibitors. All data are presented as the means ± SEMs from at least three independent experiments. *p

    Article Snippet: Briefly, RNA was extracted following a standard TRIzol protocol, and real-time PCR was performed with the ABI Step One System (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix Ex Taq II kit (TaKaRa Bio, Inc.).

    Techniques: Binding Assay, Luciferase, Transfection, Activity Assay, Western Blot, Lysis, Expressing, Real-time Polymerase Chain Reaction, Mutagenesis

    miR-210, NR1D2 and IL-6 expression in mice with cryptorchidism. ( A ) miR-210 expression in mice with cryptorchidism. ( B ) NR1D2 expression in mice with cryptorchidism. ( C ) IL-6 expression in mice with cryptorchidism. Tissue RNA was extracted with TRIzol and examined by real-time PCR. All data are presented as the means ± SEMs from at least three independent experiments. *p

    Journal: Scientific Reports

    Article Title: The functional and predictive roles of miR-210 in cryptorchidism

    doi: 10.1038/srep32265

    Figure Lengend Snippet: miR-210, NR1D2 and IL-6 expression in mice with cryptorchidism. ( A ) miR-210 expression in mice with cryptorchidism. ( B ) NR1D2 expression in mice with cryptorchidism. ( C ) IL-6 expression in mice with cryptorchidism. Tissue RNA was extracted with TRIzol and examined by real-time PCR. All data are presented as the means ± SEMs from at least three independent experiments. *p

    Article Snippet: Briefly, RNA was extracted following a standard TRIzol protocol, and real-time PCR was performed with the ABI Step One System (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix Ex Taq II kit (TaKaRa Bio, Inc.).

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

    Expression of SPARC in D283 medulloblastoma cells Medulloblastoma cells were transfected with plasmid containing full-length SPARC cDNA (pSPARC) or empty vehicle control (pEV) or mock (untreated) control for 36 hrs. (A) SPARC protein levels were determined in total cell lysates by western blotting analysis using SPARC specific primary antibody. GAPDH was used to confirm equal loading. (B) Total RNA was extracted using Trizol reagent, and qRT-PCR was performed for SPARC mRNA transcript level. (C) p53 protein levels were determined in total cell lysates by western blotting analysis using p53 specific primary antibody. GAPDH was used to confirm equal loading. (D) Total RNA was extracted using Trizol reagent, and qRT-PCR was performed for p53 mRNA transcript level or (E) p21 mRNA transcript level. Total protein levels were quantified by densitometric analysis as shown in the corresponding bar graph. ** P

    Journal: Genes & Cancer

    Article Title: SPARC overexpression alters microRNA expression profiles involved in tumor progression

    doi: 10.18632/genesandcancer.130

    Figure Lengend Snippet: Expression of SPARC in D283 medulloblastoma cells Medulloblastoma cells were transfected with plasmid containing full-length SPARC cDNA (pSPARC) or empty vehicle control (pEV) or mock (untreated) control for 36 hrs. (A) SPARC protein levels were determined in total cell lysates by western blotting analysis using SPARC specific primary antibody. GAPDH was used to confirm equal loading. (B) Total RNA was extracted using Trizol reagent, and qRT-PCR was performed for SPARC mRNA transcript level. (C) p53 protein levels were determined in total cell lysates by western blotting analysis using p53 specific primary antibody. GAPDH was used to confirm equal loading. (D) Total RNA was extracted using Trizol reagent, and qRT-PCR was performed for p53 mRNA transcript level or (E) p21 mRNA transcript level. Total protein levels were quantified by densitometric analysis as shown in the corresponding bar graph. ** P

    Article Snippet: Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from D283 mock or pEV control and pSPARC cells using TRIzol® solution (Invitrogen, Carlsbad, CA) as per standard protocol.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR

    Toll-like receptors/dsRNA complex inhibitor can decrease the viral loads of IAV in P815 cells . P815 cells treatment with TLR3/dsRNA complex inhibitor were infected with H1N1, H5N1, and H7N2 at a MOI of 0.1, exposed to LE-PolyI:C, or mock treated. Cells were homogenized in Trizol and relative viral NS gene quantification was determined by real time PCR (A) . Culture supernatants were collected at the indicated times post-infection, and virus titers in the supernatants were determined by plaque assay (B) . Results shown are pooled from three independent repeats. Asterisks indicate statistically significant increases compared to mock treatment ( ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Influenza A Viruses Replicate Productively in Mouse Mastocytoma Cells (P815) and Trigger Pro-inflammatory Cytokine and Chemokine Production through TLR3 Signaling Pathway

    doi: 10.3389/fmicb.2016.02130

    Figure Lengend Snippet: Toll-like receptors/dsRNA complex inhibitor can decrease the viral loads of IAV in P815 cells . P815 cells treatment with TLR3/dsRNA complex inhibitor were infected with H1N1, H5N1, and H7N2 at a MOI of 0.1, exposed to LE-PolyI:C, or mock treated. Cells were homogenized in Trizol and relative viral NS gene quantification was determined by real time PCR (A) . Culture supernatants were collected at the indicated times post-infection, and virus titers in the supernatants were determined by plaque assay (B) . Results shown are pooled from three independent repeats. Asterisks indicate statistically significant increases compared to mock treatment ( ∗ P

    Article Snippet: Real-Time Quantitative PCR Total RNA was extracted from cells in Trizol reagents (Invitrogen, Carlsbad, CA, USA).

    Techniques: Infection, Real-time Polymerase Chain Reaction, Plaque Assay

    Upregulation of CCL2/CCR2 is responsible for the IL-10-induced lung fibrocyte recruitment. A : BAL cells harvested from control and IL-10 OE mice were allowed to adhere for 2 h at 37°C and then, after removal of suspended cells, were subjected to total RNA preparation and Q-PCR analysis for CCR2, CCR4, and CXCR4 gene expression. B : whole lung total RNA was extracted by Trizol method and was subjected to Q-PCR analysis for CCL2, CCL19, CCL21b, and CXCL12 gene expression. C : CCL2 levels in the BAL fluids were measured by ELISA analysis. Results are ± SE of 4 animals and represent 2 independent experiments of a total number of 9–10 individual animals per group.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: New concepts of IL-10-induced lung fibrosis: fibrocyte recruitment and M2 activation in a CCL2/CCR2 axis

    doi: 10.1152/ajplung.00122.2010

    Figure Lengend Snippet: Upregulation of CCL2/CCR2 is responsible for the IL-10-induced lung fibrocyte recruitment. A : BAL cells harvested from control and IL-10 OE mice were allowed to adhere for 2 h at 37°C and then, after removal of suspended cells, were subjected to total RNA preparation and Q-PCR analysis for CCR2, CCR4, and CXCR4 gene expression. B : whole lung total RNA was extracted by Trizol method and was subjected to Q-PCR analysis for CCL2, CCL19, CCL21b, and CXCL12 gene expression. C : CCL2 levels in the BAL fluids were measured by ELISA analysis. Results are ± SE of 4 animals and represent 2 independent experiments of a total number of 9–10 individual animals per group.

    Article Snippet: Total RNA was isolated from cultured BAL cells or whole lung tissue with the Trizol method (Invitrogen Life Technologies) according to the manufacturer's protocol.

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

    (A) Multiple eIF4GI-EGFP fusion proteins are translated from a dicistronic construct in vivo. 293T cells were transfected with the indicated DNA constructs as described in Materials and Methods. Lysates were immunoprecipitated with a monoclonal anti-EGFP antibody, subjected to SDS-PAGE, and transferred to nitrocellulose. Western blotting was performed with polyclonal anti-EGFP antibodies. Lane a contains lysate from 2 × 10 6 transfected cells, while lanes b and c contain lysate from 10 7 cells. Relative luciferase activity from cell lysates is shown below the lane for the given transfected plasmid. (B) Northern blot analysis of transfected cells. Cells transfected with the indicated constructs were lysed in Trizol reagent, and RNA was isolated and run on a 1% agarose formaldehyde gel. RNA was then transferred to a nylon membrane and probed with a [ 32 P]GTP-labeled antisense EGFP riboprobe. Migration of marker RNAs is shown on the left.

    Journal: Molecular and Cellular Biology

    Article Title: Generation of Multiple Isoforms of Eukaryotic Translation Initiation Factor 4GI by Use of Alternate Translation Initiation Codons

    doi: 10.1128/MCB.22.13.4499-4511.2002

    Figure Lengend Snippet: (A) Multiple eIF4GI-EGFP fusion proteins are translated from a dicistronic construct in vivo. 293T cells were transfected with the indicated DNA constructs as described in Materials and Methods. Lysates were immunoprecipitated with a monoclonal anti-EGFP antibody, subjected to SDS-PAGE, and transferred to nitrocellulose. Western blotting was performed with polyclonal anti-EGFP antibodies. Lane a contains lysate from 2 × 10 6 transfected cells, while lanes b and c contain lysate from 10 7 cells. Relative luciferase activity from cell lysates is shown below the lane for the given transfected plasmid. (B) Northern blot analysis of transfected cells. Cells transfected with the indicated constructs were lysed in Trizol reagent, and RNA was isolated and run on a 1% agarose formaldehyde gel. RNA was then transferred to a nylon membrane and probed with a [ 32 P]GTP-labeled antisense EGFP riboprobe. Migration of marker RNAs is shown on the left.

    Article Snippet: Transfected 293 cells were lysed directly into Trizol extraction reagent (Invitrogen).

    Techniques: Construct, In Vivo, Transfection, Immunoprecipitation, SDS Page, Western Blot, Luciferase, Activity Assay, Plasmid Preparation, Northern Blot, Isolation, Labeling, Migration, Marker

    Upregulation of miR-421 in gastric cancer tissue specimens. Total RNA was isolated from gastric cancer tissues and matched adjacent normal gastric tissue using TRIzol reagent. Levels of miR-421 expression were measured using reverse transcription-quantitative

    Journal: Experimental and Therapeutic Medicine

    Article Title: MicroRNA-421 promotes the proliferation and metastasis of gastric cancer cells by targeting claudin-11

    doi: 10.3892/etm.2017.4798

    Figure Lengend Snippet: Upregulation of miR-421 in gastric cancer tissue specimens. Total RNA was isolated from gastric cancer tissues and matched adjacent normal gastric tissue using TRIzol reagent. Levels of miR-421 expression were measured using reverse transcription-quantitative

    Article Snippet: Total RNA was isolated using TRIzol® isolation reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

    Techniques: Isolation, Expressing

    Survivin expression and oncolytic adenovirus replicative activity in cell lines examined (A) Cultured gastric cancer and normal cells were collected, and the TRIzol method was used to extract the total RNA from these cells. Survivin was amplified from the extracted RNA through RT-PCR with GAPDH as the internal control. (B) Fluorescence microscopy was used to observe EGFP-positive gastric cancer cells and normal cells 48 h after the cells were infected with AdSurp-EGFP or AdCMV-EGFP at an MOI of 1 pfu/cell, original magnification 200×. (C) Gastric cancer and normal cells were infected with AdSurp-Hsp70 or AdCMV-Hsp70 at an MOI of 1 pfu/cell. Forty-eight hours after infection, the cells were collected and the virus titer was quantified using the TCID 50 assay. ** P

    Journal: Oncotarget

    Article Title: Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy

    doi:

    Figure Lengend Snippet: Survivin expression and oncolytic adenovirus replicative activity in cell lines examined (A) Cultured gastric cancer and normal cells were collected, and the TRIzol method was used to extract the total RNA from these cells. Survivin was amplified from the extracted RNA through RT-PCR with GAPDH as the internal control. (B) Fluorescence microscopy was used to observe EGFP-positive gastric cancer cells and normal cells 48 h after the cells were infected with AdSurp-EGFP or AdCMV-EGFP at an MOI of 1 pfu/cell, original magnification 200×. (C) Gastric cancer and normal cells were infected with AdSurp-Hsp70 or AdCMV-Hsp70 at an MOI of 1 pfu/cell. Forty-eight hours after infection, the cells were collected and the virus titer was quantified using the TCID 50 assay. ** P

    Article Snippet: The total RNA was extracted from the cells using the TRIzol reagent kit (Invitrogen Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer's instructions.

    Techniques: Expressing, Activity Assay, Cell Culture, Amplification, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Microscopy, Infection

    Effect of THSG on klotho expression in SHRs. Total RNA was extracted from the rat cerebral cortex, hippocampus, testis, and renal medulla by using Invitrogen's TRIzol reagent. Klotho mRNA expression was measured using the Maxima SYBR Green qPCR Master Mix. Data were normalized to GAPDH mRNA expression levels (each group n = 8). Data are expressed as mean ± SD; ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside Promotes Expression of the Longevity Gene Klotho

    doi: 10.1155/2016/3128235

    Figure Lengend Snippet: Effect of THSG on klotho expression in SHRs. Total RNA was extracted from the rat cerebral cortex, hippocampus, testis, and renal medulla by using Invitrogen's TRIzol reagent. Klotho mRNA expression was measured using the Maxima SYBR Green qPCR Master Mix. Data were normalized to GAPDH mRNA expression levels (each group n = 8). Data are expressed as mean ± SD; ∗ P

    Article Snippet: RNA Isolation and Quantitative Polymerase Chain Reaction Total RNA was extracted from rat tissues or NRK-52E cells by using Invitrogen's TRIzol reagent (Life Technologies, Grand Island, USA) according to the manufacturer's instructions.

    Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction