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Roche ribonucleic acid rna
Ribonucleic Acid Rna, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ribonucleic acid rna - by Bioz Stars, 2020-04
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Diagnostic Assay:

Article Title: Treatment of Middle East Respiratory Syndrome with a combination of lopinavir-ritonavir and interferon-β1b (MIRACLE trial): study protocol for a randomized controlled trial
Article Snippet: MERS-CoV RT-PCR testing Diagnostic RT-PCR will be preformed per each hospital protocol in its own hospital laboratory or in the reference laboratory of the Saudi Ministry of Health. .. The testing procedure includes extracting ribonucleic acid (RNA) from respiratory and serum specimens using the MagNA Pure 96 Viral NA Kit (Roche Applied Science, Indianapolis, IN, USA).

Real-time Polymerase Chain Reaction:

Article Title: Scaling up antiretroviral therapy for HIV-infected children in C?te d'Ivoire: determinants of survival and loss to programme
Article Snippet: .. Children aged < 18 months were diagnosed virologically using a TaqMan HIV-1, ribonucleic acid (RNA), real-time polymerase chain reaction test (Hoffmann–La Roche, Basel, Switzerland) with a threshold of 300 copies/ml. ..

Amplification:

Article Title: Treatment of Middle East Respiratory Syndrome with a combination of lopinavir-ritonavir and interferon-β1b (MIRACLE trial): study protocol for a randomized controlled trial
Article Snippet: The testing procedure includes extracting ribonucleic acid (RNA) from respiratory and serum specimens using the MagNA Pure 96 Viral NA Kit (Roche Applied Science, Indianapolis, IN, USA). .. We will document cycle thresholds (Ct ) for upE and ORF1a which indicate the start of exponential amplification.

Article Title: A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting
Article Snippet: Then, ribonucleic acid (RNA) was extracted using a Pure RNA Isolation Kit (Roche, Nutley, NJ, USA), and complementary DNA (cDNA) was synthesized via a Revert Aid First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). .. The amplified VHH fragments were ligated into the phagemid vector pCANTAB5E after being digested with the Sfi I restriction enzyme.

Positive Control:

Article Title: Treatment of Middle East Respiratory Syndrome with a combination of lopinavir-ritonavir and interferon-β1b (MIRACLE trial): study protocol for a randomized controlled trial
Article Snippet: The testing procedure includes extracting ribonucleic acid (RNA) from respiratory and serum specimens using the MagNA Pure 96 Viral NA Kit (Roche Applied Science, Indianapolis, IN, USA). .. A positive control for ORF1a and upE rRT-PCR is performed according to the manufacturer’s instructions.

Synthesized:

Article Title: A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting
Article Snippet: .. Then, ribonucleic acid (RNA) was extracted using a Pure RNA Isolation Kit (Roche, Nutley, NJ, USA), and complementary DNA (cDNA) was synthesized via a Revert Aid First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). .. Nested polymerase chain reaction was performed to amplify the VHH fragments by using primers specific for HcAbs, which were designed according to the constant region (CH 2) of alpaca HcAbs.

Isolation:

Article Title: Short-Term High-Starch, Low-Protein Diet Induces Reversible Increase in β-cell Mass Independent of Body Weight Gain in Mice
Article Snippet: .. Isolation of Ribonucleic Acid (RNA) and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Pancreata were perfused with collagenase 4.5 mg (cat.112490020001; Roche, Basel, Switzerland) in 3 mL RPMI medium (1×)1640 (11875-093; gibcl, Tokyo, Japan). ..

Article Title: A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting
Article Snippet: .. Then, ribonucleic acid (RNA) was extracted using a Pure RNA Isolation Kit (Roche, Nutley, NJ, USA), and complementary DNA (cDNA) was synthesized via a Revert Aid First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). .. Nested polymerase chain reaction was performed to amplify the VHH fragments by using primers specific for HcAbs, which were designed according to the constant region (CH 2) of alpaca HcAbs.

Northern Blot:

Article Title: Scaling up antiretroviral therapy for HIV-infected children in C?te d'Ivoire: determinants of survival and loss to programme
Article Snippet: In those aged ≥ 18 months, the standard serum testing algorithm comprised a series of two rapid HIV assays: the Determine® HIV-1/2 assay (Inverness Medical, Bedford, United Kingdom of Great Britain and Northern Ireland) followed by the Genie II® HIV-1/HIV-2 assay (Bio-Rad laboratories, Marne-La-Coquette, France). .. Children aged < 18 months were diagnosed virologically using a TaqMan HIV-1, ribonucleic acid (RNA), real-time polymerase chain reaction test (Hoffmann–La Roche, Basel, Switzerland) with a threshold of 300 copies/ml.

Injection:

Article Title: A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting
Article Snippet: Blood was collected from the jugular vein prior to each injection, and the sera were used to monitor the immunization process, determined by indirect ELISA. .. Then, ribonucleic acid (RNA) was extracted using a Pure RNA Isolation Kit (Roche, Nutley, NJ, USA), and complementary DNA (cDNA) was synthesized via a Revert Aid First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania).

Quantitative RT-PCR:

Article Title: Treatment of Middle East Respiratory Syndrome with a combination of lopinavir-ritonavir and interferon-β1b (MIRACLE trial): study protocol for a randomized controlled trial
Article Snippet: The testing procedure includes extracting ribonucleic acid (RNA) from respiratory and serum specimens using the MagNA Pure 96 Viral NA Kit (Roche Applied Science, Indianapolis, IN, USA). .. The extracted nucleic acids will be tested by rRT-PCR targeting the upstream MERS-CoV envelope protein gene (upE) and open-reading frame 1a (ORF1a) regions of the MERS-CoV genome on a LightCycler 480 System (Roche Diagnostics, Mannheim, Germany) [ ].

Indirect ELISA:

Article Title: A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting
Article Snippet: Blood was collected from the jugular vein prior to each injection, and the sera were used to monitor the immunization process, determined by indirect ELISA. .. Then, ribonucleic acid (RNA) was extracted using a Pure RNA Isolation Kit (Roche, Nutley, NJ, USA), and complementary DNA (cDNA) was synthesized via a Revert Aid First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Short-Term High-Starch, Low-Protein Diet Induces Reversible Increase in β-cell Mass Independent of Body Weight Gain in Mice
Article Snippet: .. Isolation of Ribonucleic Acid (RNA) and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Pancreata were perfused with collagenase 4.5 mg (cat.112490020001; Roche, Basel, Switzerland) in 3 mL RPMI medium (1×)1640 (11875-093; gibcl, Tokyo, Japan). ..

Article Title: Successful Cognitive Aging and Health-Related Quality of Life in Younger and Older Adults Infected with HIV
Article Snippet: .. To measure levels of ribonucleic acid (RNA) in plasma and cerebrospinal fluid, reverse transcriptase-polymerase chain reaction was used (RT-PCR; Amplicor, Roche Diagnostics, Indianapolis, IN). .. The Medical Outcome Study 36 Item Short-Form version 1.0 (MOS-SF-36) was used to assess HRQoL, and the reliability and construct validity of the SF-36 has been well established for use in people with HIV infection , .

Article Title: Treatment of Middle East Respiratory Syndrome with a combination of lopinavir-ritonavir and interferon-β1b (MIRACLE trial): study protocol for a randomized controlled trial
Article Snippet: Paragraph title: MERS-CoV RT-PCR testing ... The testing procedure includes extracting ribonucleic acid (RNA) from respiratory and serum specimens using the MagNA Pure 96 Viral NA Kit (Roche Applied Science, Indianapolis, IN, USA).

Infection:

Article Title: Scaling up antiretroviral therapy for HIV-infected children in C?te d'Ivoire: determinants of survival and loss to programme
Article Snippet: Children aged < 18 months were diagnosed virologically using a TaqMan HIV-1, ribonucleic acid (RNA), real-time polymerase chain reaction test (Hoffmann–La Roche, Basel, Switzerland) with a threshold of 300 copies/ml. .. All children with a confirmed HIV infection were seen monthly and had unrestricted free access to antiretroviral drugs and comprehensive care.

Nested PCR:

Article Title: A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting
Article Snippet: Then, ribonucleic acid (RNA) was extracted using a Pure RNA Isolation Kit (Roche, Nutley, NJ, USA), and complementary DNA (cDNA) was synthesized via a Revert Aid First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). .. Nested polymerase chain reaction was performed to amplify the VHH fragments by using primers specific for HcAbs, which were designed according to the constant region (CH 2) of alpaca HcAbs.

Transformation Assay:

Article Title: A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting
Article Snippet: Then, ribonucleic acid (RNA) was extracted using a Pure RNA Isolation Kit (Roche, Nutley, NJ, USA), and complementary DNA (cDNA) was synthesized via a Revert Aid First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). .. Next, the ligated vector was subsequently transformed into TG1 E. coli cells.

Chloramphenicol Acetyltransferase Assay:

Article Title: Short-Term High-Starch, Low-Protein Diet Induces Reversible Increase in β-cell Mass Independent of Body Weight Gain in Mice
Article Snippet: .. Isolation of Ribonucleic Acid (RNA) and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Pancreata were perfused with collagenase 4.5 mg (cat.112490020001; Roche, Basel, Switzerland) in 3 mL RPMI medium (1×)1640 (11875-093; gibcl, Tokyo, Japan). ..

Plasmid Preparation:

Article Title: A novel nanobody specific for respiratory surfactant protein A has potential for lung targeting
Article Snippet: Then, ribonucleic acid (RNA) was extracted using a Pure RNA Isolation Kit (Roche, Nutley, NJ, USA), and complementary DNA (cDNA) was synthesized via a Revert Aid First-Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). .. The amplified VHH fragments were ligated into the phagemid vector pCANTAB5E after being digested with the Sfi I restriction enzyme.

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  • rna  (Roche)
    96
    Roche rna
    Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si <t>RNA</t> ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative <t>PCR</t> using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P
    Rna, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna/product/Roche
    Average 96 stars, based on 175 article reviews
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    rna - by Bioz Stars, 2020-04
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    93
    Roche dnmt3a sirna cagugguguguguugagaatt
    DNA methyltransferase (DNMT)3A methylated pre‐micro RNA (miR)‐145 and downregulated miR‐145. A, Real‐time PCR shows that expression of miR‐145 was increased in SKOV3 and 3AO cell lines after treatment with 5‐aza‐2′‐deoxycytidine (5‐Aza‐CdR) for 5 days compared with mock‐treated cells. B, Quantitative real‐time PCR (qRT‐PCR) results show that <t>DNMT3A</t> mRNA level was significantly reduced by siDNMT3A transfection. C, qRT‐PCR shows that knockdown of DNMT3A increased miR‐145 in both SKOV3 and 3AO cells. D, Western blot assays show that knockdown of DNMT3A decreased DNMT3A expression. E, Quantitative analysis of methylation‐specific PCR (MSP) results show that the methylated proportion of miR‐145 precursor gene in DNMT3A <t>siRNA‐transfected</t> cells was lower than in control cells. F, qRT‐PCR results show that DNMT3A mRNA level was significantly increased by pcDNA3/Myc‐DNMT3A transfection. G, qRT‐PCR shows that overexpression of DNMT3A decreased miR‐145 in both SKOV3 and 3AO cells. H, Western blot assays show that overexpression of DNMT3A increased DNMT3A expression. I, Quantitative analysis of MSP results show that the methylated proportion of miR‐145 precursor gene in pcDNA3/Myc‐DNMT3A‐transfected cells was higher than in control cells. P
    Dnmt3a Sirna Cagugguguguguugagaatt, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnmt3a sirna cagugguguguguugagaatt/product/Roche
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    92
    Roche qrt pcr analysis total rna
    Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by <t>qRT-PCR.</t> Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p
    Qrt Pcr Analysis Total Rna, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
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    Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si RNA ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative PCR using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P

    Journal: Cancer Science

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1

    doi: 10.1111/cas.13754

    Figure Lengend Snippet: Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si RNA ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative PCR using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P

    Article Snippet: 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Construct, Chromatin Immunoprecipitation, Over Expression, Activity Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

    T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

    Journal: Genome Medicine

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    doi: 10.1186/s13073-018-0589-3

    Figure Lengend Snippet: T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

    Article Snippet: Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche).

    Techniques: Activation Assay, Transformation Assay, Purification, Selection, RNA Sequencing Assay, Chromatin Immunoprecipitation, Sequencing, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Activity Assay, In Vitro, Expressing, Quantitative RT-PCR, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Positive Control, Staining, FACS

    DNA methyltransferase (DNMT)3A methylated pre‐micro RNA (miR)‐145 and downregulated miR‐145. A, Real‐time PCR shows that expression of miR‐145 was increased in SKOV3 and 3AO cell lines after treatment with 5‐aza‐2′‐deoxycytidine (5‐Aza‐CdR) for 5 days compared with mock‐treated cells. B, Quantitative real‐time PCR (qRT‐PCR) results show that DNMT3A mRNA level was significantly reduced by siDNMT3A transfection. C, qRT‐PCR shows that knockdown of DNMT3A increased miR‐145 in both SKOV3 and 3AO cells. D, Western blot assays show that knockdown of DNMT3A decreased DNMT3A expression. E, Quantitative analysis of methylation‐specific PCR (MSP) results show that the methylated proportion of miR‐145 precursor gene in DNMT3A siRNA‐transfected cells was lower than in control cells. F, qRT‐PCR results show that DNMT3A mRNA level was significantly increased by pcDNA3/Myc‐DNMT3A transfection. G, qRT‐PCR shows that overexpression of DNMT3A decreased miR‐145 in both SKOV3 and 3AO cells. H, Western blot assays show that overexpression of DNMT3A increased DNMT3A expression. I, Quantitative analysis of MSP results show that the methylated proportion of miR‐145 precursor gene in pcDNA3/Myc‐DNMT3A‐transfected cells was higher than in control cells. P

    Journal: Cancer Science

    Article Title: Double‐negative feedback interaction between DNA methyltransferase 3A and microRNA‐145 in the Warburg effect of ovarian cancer cells. Double‐negative feedback interaction between DNA methyltransferase 3A and microRNA‐145 in the Warburg effect of ovarian cancer cells

    doi: 10.1111/cas.13734

    Figure Lengend Snippet: DNA methyltransferase (DNMT)3A methylated pre‐micro RNA (miR)‐145 and downregulated miR‐145. A, Real‐time PCR shows that expression of miR‐145 was increased in SKOV3 and 3AO cell lines after treatment with 5‐aza‐2′‐deoxycytidine (5‐Aza‐CdR) for 5 days compared with mock‐treated cells. B, Quantitative real‐time PCR (qRT‐PCR) results show that DNMT3A mRNA level was significantly reduced by siDNMT3A transfection. C, qRT‐PCR shows that knockdown of DNMT3A increased miR‐145 in both SKOV3 and 3AO cells. D, Western blot assays show that knockdown of DNMT3A decreased DNMT3A expression. E, Quantitative analysis of methylation‐specific PCR (MSP) results show that the methylated proportion of miR‐145 precursor gene in DNMT3A siRNA‐transfected cells was lower than in control cells. F, qRT‐PCR results show that DNMT3A mRNA level was significantly increased by pcDNA3/Myc‐DNMT3A transfection. G, qRT‐PCR shows that overexpression of DNMT3A decreased miR‐145 in both SKOV3 and 3AO cells. H, Western blot assays show that overexpression of DNMT3A increased DNMT3A expression. I, Quantitative analysis of MSP results show that the methylated proportion of miR‐145 precursor gene in pcDNA3/Myc‐DNMT3A‐transfected cells was higher than in control cells. P

    Article Snippet: DNMT3A siRNA (CAGUGGUGUGUGUUGAGAATT) was transiently transfected 100 nmol/L per well using the X‐treme GENE siRNA Transfection Reagent (Roche).

    Techniques: Methylation, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Polymerase Chain Reaction, Over Expression

    Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by qRT-PCR. Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p

    Journal: International Journal of Molecular Sciences

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice

    doi: 10.3390/ijms19061786

    Figure Lengend Snippet: Quercetin increases uncoupling protein-1 ( Ucp1) gene expression specifically in subcutaneous white adipose tissue. Gene expression in sWAT ( A ) and BAT ( B ) was determined by qRT-PCR. Hematoxylin and eosin (H E) staining was performed on paraffin-embedded sWAT sections, and representative pictures are shown ( C ). Pictures were analyzed in ImageJ to determine the relative cell size ( D ). sWAT sections were stained for UCP-1 (arrows indicate UCP-1 positive cells, E ) as well. BAT sections were stained for H E ( F ) and used to quantify lipid droplet content in ImageJ ( G ). BAT sections were also stained for UCP-1 ( H ). Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β 2-microglobulin (sWAT), Gapdh , and Hprt (BAT), * p

    Article Snippet: RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands).

    Techniques: Expressing, Quantitative RT-PCR, Staining

    Quercetin reduces hepatic apolipoprotein B ( Apob) expression and increases the uptake of triglycerides (TG)-derived fatty acid (FA) by subcutaneous white adipose tissue. In week 2 and week 10 of the intervention, 24 h feces was collected ( A ) and used to determine fecal free fatty acid (FFA) concentration ( B ). Gene expression in the liver was determined by qRT-PCR for acyl-CoA synthetase long-chain family member 1 ( Acsl1) , acetyl-CoA carboxylase 2 ( Acc2 ), microsomal triglyceride transfer protein ( Mttp ), and Apob ( C ). After 12 weeks, mice were injected with glycerol tri[ 3 H]oleate-labeled lipoprotein-like particles, and clearance from plasma ( D ) and uptake per gram organ ( E ) were determined by 3 H-activity analysis. Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β2-microglobulin , * p

    Journal: International Journal of Molecular Sciences

    Article Title: Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice

    doi: 10.3390/ijms19061786

    Figure Lengend Snippet: Quercetin reduces hepatic apolipoprotein B ( Apob) expression and increases the uptake of triglycerides (TG)-derived fatty acid (FA) by subcutaneous white adipose tissue. In week 2 and week 10 of the intervention, 24 h feces was collected ( A ) and used to determine fecal free fatty acid (FFA) concentration ( B ). Gene expression in the liver was determined by qRT-PCR for acyl-CoA synthetase long-chain family member 1 ( Acsl1) , acetyl-CoA carboxylase 2 ( Acc2 ), microsomal triglyceride transfer protein ( Mttp ), and Apob ( C ). After 12 weeks, mice were injected with glycerol tri[ 3 H]oleate-labeled lipoprotein-like particles, and clearance from plasma ( D ) and uptake per gram organ ( E ) were determined by 3 H-activity analysis. Data are represented as mean ± SEM ( n = 8–10); the expression of genes was corrected for the reference gene β2-microglobulin , * p

    Article Snippet: RNA Isolation and qRT-PCR Analysis Total RNA was isolated using TriPure Isolation reagent (Roche obtained via Sigma, St. Louis, MO, USA ) following the manufacturer’s protocol. cDNA was made using Moloney Murine Leukemia Virus Reverse Transcriptase (Promega, Leiden, The Netherlands).

    Techniques: Expressing, Derivative Assay, Concentration Assay, Quantitative RT-PCR, Mouse Assay, Injection, Labeling, Activity Assay