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Molecular Research Center inc total ribonucleic acid rna
Total Ribonucleic Acid Rna, supplied by Molecular Research Center inc, 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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Average 93 stars, based on 1 article reviews
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Real-time Polymerase Chain Reaction:

Article Title: Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells
Article Snippet: .. Quantitative reverse transcription polymerase chain reaction (qPCR) for analysis of eNOS mRNA expression After treatment for 24 hours, total ribonucleic acid (RNA) from HUVECs was extracted using TRI Reagent (Molecular Research Center, Cincinnati, USA) according to a previously published protocol. .. Polyacryl carrier (Molecular Research Center, Cincinnati, USA) was added to precipitate the total RNA.

Article Title: Sildenafil ameliorates right ventricular early molecular derangement during left ventricular pressure overload
Article Snippet: Paragraph title: Quantitative real-time polymerase chain reaction (PCR) ... Total ribonucleic acid (RNA) was extracted from frozen heart tissue using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions.

Cell Isolation:

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: Paragraph title: Murine T cell isolation and differentiation ... Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells
Article Snippet: .. Quantitative reverse transcription polymerase chain reaction (qPCR) for analysis of eNOS mRNA expression After treatment for 24 hours, total ribonucleic acid (RNA) from HUVECs was extracted using TRI Reagent (Molecular Research Center, Cincinnati, USA) according to a previously published protocol. .. Polyacryl carrier (Molecular Research Center, Cincinnati, USA) was added to precipitate the total RNA.

Magnetic Beads:

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: Naïve cells were collected from this population by selecting for CD4+ CD62Lhigh CD25low CD44low cells ( > 97% purity; Dako Cytomation, Glostrup, Denmark) and were also obtained using CD4+ CD62L+ magnetic beads (Miltenyi Biotec). .. Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

Isolation:

Article Title: Crohn’s Disease and Ulcerative Colitis Show Unique Cytokine Profiles
Article Snippet: .. Protein and total ribonucleic acid (RNA) were isolated from specimens using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) following manufacturer’s instructions. .. Protein and RNA concentration were measured using BMG Labtec FLUOstar OPTIMA microplate reader (BMG LABTECH Inc., Cary, NC, USA).

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: To purify splenic CD4+ T cells, splenocytes were incubated with CD4-coated magnetic beads and isolated on magnetic-activated cell sorting (MACS) separation columns (Miltenyi Biotec, Seoul, South Korea). .. Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

Derivative Assay:

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: To induce Th17-polarization, splenocytes were stimulated with plate-bound anti-CD3 (145-2C11) (0.5 μg/ml; BD Pharmingen, San Jose, CA), anti-CD28 (37.51) (1 μg/ml; eBiosciences, San Diego, CA), anti-interferon-γ (37895) (IFN-γ; 2 μg/ml), anti-IL-4 (30340) (2 μg/ml), recombinant human (rh) TGF-β (CHO cell derived) (2 ng/ml; all from Pepro-Tech, Rocky Hill, NJ), and IL-6 (E.coli derived) (20 ng/ml; R & D Systems, Minneapolis, MN) for 72 h. Th0 cells were stimulated with only anti-CD3 and anti-CD28, in the absence of added cytokines. .. Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

Spectrophotometry:

Article Title: Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells
Article Snippet: Quantitative reverse transcription polymerase chain reaction (qPCR) for analysis of eNOS mRNA expression After treatment for 24 hours, total ribonucleic acid (RNA) from HUVECs was extracted using TRI Reagent (Molecular Research Center, Cincinnati, USA) according to a previously published protocol. .. The extracted total RNA was assessed for its purity and quantity using a Nanodrop ND-100 spectrophotometer (Wilmington DE, USA) and stored at −80 °C.

Mouse Assay:

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: Murine T cell isolation and differentiation Mouse spleen cells were obtained from naïve DBA/1J mice and sieved through a mesh and red blood cells (RBCs) lysed in hypotonic ACK buffer (0.15 mM NH4 Cl, 1 mM KCO3 , and 0.1 mM EDTA; pH 7.4). .. Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

SYBR Green Assay:

Article Title: Sildenafil ameliorates right ventricular early molecular derangement during left ventricular pressure overload
Article Snippet: Total ribonucleic acid (RNA) was extracted from frozen heart tissue using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. .. Quantitative real-time PCR was carried out using SYBR Green assay or Taqman probe assay.

Concentration Assay:

Article Title: Crohn’s Disease and Ulcerative Colitis Show Unique Cytokine Profiles
Article Snippet: Protein and total ribonucleic acid (RNA) were isolated from specimens using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) following manufacturer’s instructions. .. Protein and RNA concentration were measured using BMG Labtec FLUOstar OPTIMA microplate reader (BMG LABTECH Inc., Cary, NC, USA).

Incubation:

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: To purify splenic CD4+ T cells, splenocytes were incubated with CD4-coated magnetic beads and isolated on magnetic-activated cell sorting (MACS) separation columns (Miltenyi Biotec, Seoul, South Korea). .. Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

Cell Culture:

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: Cells were cultured at RPMI (5% FBS, 1% antibiotics). .. Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

Expressing:

Article Title: Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells
Article Snippet: .. Quantitative reverse transcription polymerase chain reaction (qPCR) for analysis of eNOS mRNA expression After treatment for 24 hours, total ribonucleic acid (RNA) from HUVECs was extracted using TRI Reagent (Molecular Research Center, Cincinnati, USA) according to a previously published protocol. .. Polyacryl carrier (Molecular Research Center, Cincinnati, USA) was added to precipitate the total RNA.

Polymerase Chain Reaction:

Article Title: Sildenafil ameliorates right ventricular early molecular derangement during left ventricular pressure overload
Article Snippet: Paragraph title: Quantitative real-time polymerase chain reaction (PCR) ... Total ribonucleic acid (RNA) was extracted from frozen heart tissue using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions.

FACS:

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: To purify splenic CD4+ T cells, splenocytes were incubated with CD4-coated magnetic beads and isolated on magnetic-activated cell sorting (MACS) separation columns (Miltenyi Biotec, Seoul, South Korea). .. Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

Recombinant:

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: To induce Th17-polarization, splenocytes were stimulated with plate-bound anti-CD3 (145-2C11) (0.5 μg/ml; BD Pharmingen, San Jose, CA), anti-CD28 (37.51) (1 μg/ml; eBiosciences, San Diego, CA), anti-interferon-γ (37895) (IFN-γ; 2 μg/ml), anti-IL-4 (30340) (2 μg/ml), recombinant human (rh) TGF-β (CHO cell derived) (2 ng/ml; all from Pepro-Tech, Rocky Hill, NJ), and IL-6 (E.coli derived) (20 ng/ml; R & D Systems, Minneapolis, MN) for 72 h. Th0 cells were stimulated with only anti-CD3 and anti-CD28, in the absence of added cytokines. .. Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

Magnetic Cell Separation:

Article Title: Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells, via Inhibition of NF-κB
Article Snippet: To purify splenic CD4+ T cells, splenocytes were incubated with CD4-coated magnetic beads and isolated on magnetic-activated cell sorting (MACS) separation columns (Miltenyi Biotec, Seoul, South Korea). .. Total ribonucleic acid (RNA) was extracted from splenocytes by using the Tri reagent (Molecular Research Center, Cincinnati, OH).

TaqMan Probe Assay:

Article Title: Sildenafil ameliorates right ventricular early molecular derangement during left ventricular pressure overload
Article Snippet: Total ribonucleic acid (RNA) was extracted from frozen heart tissue using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. .. Quantitative real-time PCR was carried out using SYBR Green assay or Taqman probe assay.

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    Molecular Research Center inc qpcr total rna
    NHBE cells express CXCR4 but not CCR5 and HIV gp120 induces mucus through CXCR4. ( A ): NHBE cell <t>RNA</t> was analyzed by <t>qPCR</t> for the expression of CXCR4 and CCR5 up to 40 cycles of amplification. ( B ): gp120 LAV -induced mucus formation was determined in the presence or absence of CXCR4 inhibitor AMD3100 and CCR5 inhibitor maraviroc. ( C ): gp120-induced mRNA expression of MUC5AC and GABA A Rα2 was determined in the presence and absence of AMD3100 and maraviroc by qPCR analysis. Each experiment was repeated at least three times; bars represent mean ± SEM.
    Qpcr Total Rna, supplied by Molecular Research Center inc, used in various techniques. Bioz Stars score: 96/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Molecular Research Center inc rt pcr total rna
    RFP-tagged stMSC vect cells within the gastric mucosa of mice treated with IFNγ. Gastric mucosa collected from mice transplanted with stMSC vect cells injected with ( A, B ) PBS or ( C, D ) IFNγ or with stMSC ShhKO cells injected with ( E, F ) IFNγ were stained with anti-RFP antibody (brown). Higher magnification of image in ( A, C and E ) are shown in ( B, D and F ). ( G ) <t>RT-PCR</t> using <t>RNA</t> isolated from stomachs of PBS- or IFNγ-treated mice transplanted with stMSC vect or stMSC ShhKO cells. ( H ) Morphometric analysis of RFP-positive cells/gland in the stomachs of mice transplanted with wtMSC, wtMSC Shh , stMSCs vect or stMSCs ShhKO treated with rmIFNγ for 21 days. *P
    Rt Pcr Total Rna, supplied by Molecular Research Center inc, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Molecular Research Center inc quantitative rt pcr qrt pcr analysis total rna
    HuR regulates PDCD4 stability via miR-21 A . HeLa cells were transiently transfected with a miR-21 mimic for 24 h and cells were harvested for western blot analysis. Tubulin was used as a loading control. B . HeLa cells were transiently transfected with a miR-21 mimic for 24 h and <t>RNA</t> was harvested. <t>qRT-PCR</t> analysis showing decrease of PDCD4 mRNA relative to GAPDH after miR-21 over-expression. C . Left panel: AntimiR-21 or antimiR-CTRL (control) was transiently transfected into HeLa cells for 24 h followed by siHuR transfection for an additional 48 h. Cells were harvested and protein levels were analyzed by western blot. Right panel: Quantification of PDCD4 protein levels relative to Tubulin.
    Quantitative Rt Pcr Qrt Pcr Analysis Total Rna, supplied by Molecular Research Center inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Molecular Research Center inc quantitative real time pcr total rna
    Charts displaying the in vivo radial positioning and expression profile of B. glabrata actin gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni to normal miracidia (top row of charts, in red) or irradiated attenuated miracidia (bottom row of charts, in grey) over 30 minutes, 2 hours, 5 hours and 24 hours. B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH or <t>RNA</t> was isolated and <t>q-RT-PCR</t> was performed (middle chart). In the NMRI snail strain actin is repositioned within interphase nuclei after infection and this is correlated with changes in gene expression (A). No repositioning is observed in the BS-90 snails, however the gene is expressed 30 minutes after infection (B). No repositioning of gene loci or change in expression was observed for actin in snails infected with attenuated miracidia. Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P
    Quantitative Real Time Pcr Total Rna, supplied by Molecular Research Center inc, used in various techniques. Bioz Stars score: 92/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative real time pcr total rna/product/Molecular Research Center inc
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    NHBE cells express CXCR4 but not CCR5 and HIV gp120 induces mucus through CXCR4. ( A ): NHBE cell RNA was analyzed by qPCR for the expression of CXCR4 and CCR5 up to 40 cycles of amplification. ( B ): gp120 LAV -induced mucus formation was determined in the presence or absence of CXCR4 inhibitor AMD3100 and CCR5 inhibitor maraviroc. ( C ): gp120-induced mRNA expression of MUC5AC and GABA A Rα2 was determined in the presence and absence of AMD3100 and maraviroc by qPCR analysis. Each experiment was repeated at least three times; bars represent mean ± SEM.

    Journal: PLoS ONE

    Article Title: HIV gp120 Induces Mucus Formation in Human Bronchial Epithelial Cells through CXCR4/?7-Nicotinic Acetylcholine Receptors

    doi: 10.1371/journal.pone.0077160

    Figure Lengend Snippet: NHBE cells express CXCR4 but not CCR5 and HIV gp120 induces mucus through CXCR4. ( A ): NHBE cell RNA was analyzed by qPCR for the expression of CXCR4 and CCR5 up to 40 cycles of amplification. ( B ): gp120 LAV -induced mucus formation was determined in the presence or absence of CXCR4 inhibitor AMD3100 and CCR5 inhibitor maraviroc. ( C ): gp120-induced mRNA expression of MUC5AC and GABA A Rα2 was determined in the presence and absence of AMD3100 and maraviroc by qPCR analysis. Each experiment was repeated at least three times; bars represent mean ± SEM.

    Article Snippet: RT-PCR and qPCR Total RNA from human and macaque lungs and NHBE cells was isolated using TRI-Reagent (Molecular Research Center, Inc., Cincinnati, OH). qPCR was performed using Step One plus Detection System (Applied Biosystems, Foster City, CA) and TaqMan One-Step RT-PCR kit containing AmpliTaq Gold® DNA polymerase.

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

    RFP-tagged stMSC vect cells within the gastric mucosa of mice treated with IFNγ. Gastric mucosa collected from mice transplanted with stMSC vect cells injected with ( A, B ) PBS or ( C, D ) IFNγ or with stMSC ShhKO cells injected with ( E, F ) IFNγ were stained with anti-RFP antibody (brown). Higher magnification of image in ( A, C and E ) are shown in ( B, D and F ). ( G ) RT-PCR using RNA isolated from stomachs of PBS- or IFNγ-treated mice transplanted with stMSC vect or stMSC ShhKO cells. ( H ) Morphometric analysis of RFP-positive cells/gland in the stomachs of mice transplanted with wtMSC, wtMSC Shh , stMSCs vect or stMSCs ShhKO treated with rmIFNγ for 21 days. *P

    Journal: PLoS ONE

    Article Title: Sonic Hedgehog Mediates the Proliferation and Recruitment of Transformed Mesenchymal Stem Cells to the Stomach

    doi: 10.1371/journal.pone.0075225

    Figure Lengend Snippet: RFP-tagged stMSC vect cells within the gastric mucosa of mice treated with IFNγ. Gastric mucosa collected from mice transplanted with stMSC vect cells injected with ( A, B ) PBS or ( C, D ) IFNγ or with stMSC ShhKO cells injected with ( E, F ) IFNγ were stained with anti-RFP antibody (brown). Higher magnification of image in ( A, C and E ) are shown in ( B, D and F ). ( G ) RT-PCR using RNA isolated from stomachs of PBS- or IFNγ-treated mice transplanted with stMSC vect or stMSC ShhKO cells. ( H ) Morphometric analysis of RFP-positive cells/gland in the stomachs of mice transplanted with wtMSC, wtMSC Shh , stMSCs vect or stMSCs ShhKO treated with rmIFNγ for 21 days. *P

    Article Snippet: RNA Isolation and RT-PCR Total RNA was isolated from stomach tissue using Trizol reagent according to the manufacturers protocol (TriReagent, Molecular Research Center, Inc.).

    Techniques: Mouse Assay, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Isolation

    HuR regulates PDCD4 stability via miR-21 A . HeLa cells were transiently transfected with a miR-21 mimic for 24 h and cells were harvested for western blot analysis. Tubulin was used as a loading control. B . HeLa cells were transiently transfected with a miR-21 mimic for 24 h and RNA was harvested. qRT-PCR analysis showing decrease of PDCD4 mRNA relative to GAPDH after miR-21 over-expression. C . Left panel: AntimiR-21 or antimiR-CTRL (control) was transiently transfected into HeLa cells for 24 h followed by siHuR transfection for an additional 48 h. Cells were harvested and protein levels were analyzed by western blot. Right panel: Quantification of PDCD4 protein levels relative to Tubulin.

    Journal: Oncotarget

    Article Title: ERK8 is a novel HuR kinase that regulates tumour suppressor PDCD4 through a miR-21 dependent mechanism

    doi:

    Figure Lengend Snippet: HuR regulates PDCD4 stability via miR-21 A . HeLa cells were transiently transfected with a miR-21 mimic for 24 h and cells were harvested for western blot analysis. Tubulin was used as a loading control. B . HeLa cells were transiently transfected with a miR-21 mimic for 24 h and RNA was harvested. qRT-PCR analysis showing decrease of PDCD4 mRNA relative to GAPDH after miR-21 over-expression. C . Left panel: AntimiR-21 or antimiR-CTRL (control) was transiently transfected into HeLa cells for 24 h followed by siHuR transfection for an additional 48 h. Cells were harvested and protein levels were analyzed by western blot. Right panel: Quantification of PDCD4 protein levels relative to Tubulin.

    Article Snippet: RNA extraction and quantitative RT-PCR (qRT-PCR) analysis Total RNA was isolated from cells using RNAzol (Molecular Research Center, Inc.) as per manufacturer's protocol. cDNA was generated using the First-strand cDNA synthesis kit (GE Biosciences).

    Techniques: Transfection, Western Blot, Quantitative RT-PCR, Over Expression

    HuR directly binds to PDCD4 3′UTR mRNA to regulate its protein expression A . Left panel: Western blot analysis of PDCD4 protein levels after HuR knockdown. HeLa cells were treated with siHuR or siCTRL (non-targeting control) for 72 h and harvested for western blot analysis. Tubulin was used as a loading control. Right panel: PDCD4 protein levels are quantified relative to Tubulin. B . HeLa cells were treated with siHuR or siCTRL for 72 h, harvested, and total RNA was isolated. PDCD4 mRNA levels were quantified by qRT-PCR and are shown relative to GAPDH mRNA levels. C . Seventy-two hours after siRNA transfection, HeLa cells were treated with 5 μg/mL actinomycin D. After the chase period, cells were processed for qRT-PCR to determine the mRNA half-life (11.6h for siCTRL; 9.5h for siHuR). D . Top panel: HeLa cells were crosslinked with formaldehyde and endogenous HuR was immunoprecipitated with mouse anti-HuR antibody; IgG was used as a control. Western blot analysis shows the level of immunoprecipitated HuR. Bottom panel: HuR-bound RNA was isolated and quantified by qRT-PCR, and is shown relative to IgG-immunoprecipitated material. The levels of GAPDH and RPL13 in HuR immunoprecipitation were determined as specificity controls E . PDCD4 3′UTR RNA was in vitro transcribed, 32 P labelled and UV crosslinking was performed with recombinant GST (control) or GST-HuR, separated by SDS-PAGE, and exposed to X-Ray film.

    Journal: Oncotarget

    Article Title: ERK8 is a novel HuR kinase that regulates tumour suppressor PDCD4 through a miR-21 dependent mechanism

    doi:

    Figure Lengend Snippet: HuR directly binds to PDCD4 3′UTR mRNA to regulate its protein expression A . Left panel: Western blot analysis of PDCD4 protein levels after HuR knockdown. HeLa cells were treated with siHuR or siCTRL (non-targeting control) for 72 h and harvested for western blot analysis. Tubulin was used as a loading control. Right panel: PDCD4 protein levels are quantified relative to Tubulin. B . HeLa cells were treated with siHuR or siCTRL for 72 h, harvested, and total RNA was isolated. PDCD4 mRNA levels were quantified by qRT-PCR and are shown relative to GAPDH mRNA levels. C . Seventy-two hours after siRNA transfection, HeLa cells were treated with 5 μg/mL actinomycin D. After the chase period, cells were processed for qRT-PCR to determine the mRNA half-life (11.6h for siCTRL; 9.5h for siHuR). D . Top panel: HeLa cells were crosslinked with formaldehyde and endogenous HuR was immunoprecipitated with mouse anti-HuR antibody; IgG was used as a control. Western blot analysis shows the level of immunoprecipitated HuR. Bottom panel: HuR-bound RNA was isolated and quantified by qRT-PCR, and is shown relative to IgG-immunoprecipitated material. The levels of GAPDH and RPL13 in HuR immunoprecipitation were determined as specificity controls E . PDCD4 3′UTR RNA was in vitro transcribed, 32 P labelled and UV crosslinking was performed with recombinant GST (control) or GST-HuR, separated by SDS-PAGE, and exposed to X-Ray film.

    Article Snippet: RNA extraction and quantitative RT-PCR (qRT-PCR) analysis Total RNA was isolated from cells using RNAzol (Molecular Research Center, Inc.) as per manufacturer's protocol. cDNA was generated using the First-strand cDNA synthesis kit (GE Biosciences).

    Techniques: Expressing, Western Blot, Isolation, Quantitative RT-PCR, Transfection, Immunoprecipitation, In Vitro, Recombinant, SDS Page

    H 2 O 2 causes cytoplasmic accumulation of HuR and a loss in PDCD4 expression that is mediated by miR-21 A . HuR localization by immunofluorescence of HeLa cells treated with PBS (0 mM H 2 O 2 ) or 0.5 mM H 2 O 2 for 1 h. Nuclei are visualized by Hoechst staining. Nuclear/Cytoplasmic ratio of HuR is shown on the right. Higher ratio denotes more nuclear staining. B . Left panel: HeLa cells were treated with 0.5 mM H 2 O 2 for the indicated times and cell lysates analysed by western blot analysis indicating a decrease in PDCD4 protein at 3 h as compared to Tubulin control. Right panel: PDCD4 protein levels were quantified relative to Tubulin. C . Cells were treated with 0.5 mM H 2 O 2 for the indicated time points, total RNA was isolated and analysed by qRT-PCR indicating a loss of PDCD4 mRNA as compared to GAPDH control. D . Left panel: HeLa cells were treated with antimiR-21 or a non-targeting antimiR-CTRL (control) for 24 h followed by treatment with 0.5 mM H 2 O 2 for 4 h. Cells were harvested and analysed by western blot analysis. Tubulin was used as a loading control. Right panel: Quantification of PDCD4 levels relative to Tubulin. E . HeLa cells were treated with 0.5 mM H 2 O 2 or PBS and HuR was immunoprecipitated. Bound RNA was isolated and qRT-PCR was performed to determine levels of PDCD4 mRNA. The levels of HuR-bound PDCD4 in PBS-treated cells were set as 1.

    Journal: Oncotarget

    Article Title: ERK8 is a novel HuR kinase that regulates tumour suppressor PDCD4 through a miR-21 dependent mechanism

    doi:

    Figure Lengend Snippet: H 2 O 2 causes cytoplasmic accumulation of HuR and a loss in PDCD4 expression that is mediated by miR-21 A . HuR localization by immunofluorescence of HeLa cells treated with PBS (0 mM H 2 O 2 ) or 0.5 mM H 2 O 2 for 1 h. Nuclei are visualized by Hoechst staining. Nuclear/Cytoplasmic ratio of HuR is shown on the right. Higher ratio denotes more nuclear staining. B . Left panel: HeLa cells were treated with 0.5 mM H 2 O 2 for the indicated times and cell lysates analysed by western blot analysis indicating a decrease in PDCD4 protein at 3 h as compared to Tubulin control. Right panel: PDCD4 protein levels were quantified relative to Tubulin. C . Cells were treated with 0.5 mM H 2 O 2 for the indicated time points, total RNA was isolated and analysed by qRT-PCR indicating a loss of PDCD4 mRNA as compared to GAPDH control. D . Left panel: HeLa cells were treated with antimiR-21 or a non-targeting antimiR-CTRL (control) for 24 h followed by treatment with 0.5 mM H 2 O 2 for 4 h. Cells were harvested and analysed by western blot analysis. Tubulin was used as a loading control. Right panel: Quantification of PDCD4 levels relative to Tubulin. E . HeLa cells were treated with 0.5 mM H 2 O 2 or PBS and HuR was immunoprecipitated. Bound RNA was isolated and qRT-PCR was performed to determine levels of PDCD4 mRNA. The levels of HuR-bound PDCD4 in PBS-treated cells were set as 1.

    Article Snippet: RNA extraction and quantitative RT-PCR (qRT-PCR) analysis Total RNA was isolated from cells using RNAzol (Molecular Research Center, Inc.) as per manufacturer's protocol. cDNA was generated using the First-strand cDNA synthesis kit (GE Biosciences).

    Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Isolation, Quantitative RT-PCR, Immunoprecipitation

    Charts displaying the in vivo radial positioning and expression profile of B. glabrata actin gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni to normal miracidia (top row of charts, in red) or irradiated attenuated miracidia (bottom row of charts, in grey) over 30 minutes, 2 hours, 5 hours and 24 hours. B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH or RNA was isolated and q-RT-PCR was performed (middle chart). In the NMRI snail strain actin is repositioned within interphase nuclei after infection and this is correlated with changes in gene expression (A). No repositioning is observed in the BS-90 snails, however the gene is expressed 30 minutes after infection (B). No repositioning of gene loci or change in expression was observed for actin in snails infected with attenuated miracidia. Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Differential Spatial Repositioning of Activated Genes in Biomphalaria glabrata Snails Infected with Schistosoma mansoni

    doi: 10.1371/journal.pntd.0003013

    Figure Lengend Snippet: Charts displaying the in vivo radial positioning and expression profile of B. glabrata actin gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni to normal miracidia (top row of charts, in red) or irradiated attenuated miracidia (bottom row of charts, in grey) over 30 minutes, 2 hours, 5 hours and 24 hours. B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH or RNA was isolated and q-RT-PCR was performed (middle chart). In the NMRI snail strain actin is repositioned within interphase nuclei after infection and this is correlated with changes in gene expression (A). No repositioning is observed in the BS-90 snails, however the gene is expressed 30 minutes after infection (B). No repositioning of gene loci or change in expression was observed for actin in snails infected with attenuated miracidia. Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P

    Article Snippet: Quantitative real time-PCR Total RNA from the whole body of B. glabrata adult snails (∼8–12 mm shell diameter) either unexposed (0 min) or exposed was extracted individually by RNAzol RT (Molecular Research Center, Inc.) according to the manufacturer's manual.

    Techniques: In Vivo, Expressing, Derivative Assay, Irradiation, Infection, Fluorescence In Situ Hybridization, Isolation, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    Charts displaying the in vivo radial positioning and expression profile of B. glabrata ferritin gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni normal miracidia (top row of charts, red) and attenuated irradiated miracidia (bottom row of charts, grey). B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH or RNA was isolated and a q-RT-PCR was performed (middle chart). In the NMRI strain ferritin is repositioned in snails infected with both normal and irradiated miracidia. However the gene is up-regulated only in snails infected with irradiated miracidia (A). Repositioning of ferritin gene loci is observed in the BS-90 strain snails infected with normal and irradiated miracidia, and this is correlated with up-regulation of its expression (B). Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Differential Spatial Repositioning of Activated Genes in Biomphalaria glabrata Snails Infected with Schistosoma mansoni

    doi: 10.1371/journal.pntd.0003013

    Figure Lengend Snippet: Charts displaying the in vivo radial positioning and expression profile of B. glabrata ferritin gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni normal miracidia (top row of charts, red) and attenuated irradiated miracidia (bottom row of charts, grey). B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH or RNA was isolated and a q-RT-PCR was performed (middle chart). In the NMRI strain ferritin is repositioned in snails infected with both normal and irradiated miracidia. However the gene is up-regulated only in snails infected with irradiated miracidia (A). Repositioning of ferritin gene loci is observed in the BS-90 strain snails infected with normal and irradiated miracidia, and this is correlated with up-regulation of its expression (B). Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P

    Article Snippet: Quantitative real time-PCR Total RNA from the whole body of B. glabrata adult snails (∼8–12 mm shell diameter) either unexposed (0 min) or exposed was extracted individually by RNAzol RT (Molecular Research Center, Inc.) according to the manufacturer's manual.

    Techniques: In Vivo, Expressing, Derivative Assay, Irradiation, Infection, Fluorescence In Situ Hybridization, Isolation, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    Charts displaying the in vivo radial positioning and expression profile of B. glabrata Hsp 70 gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni normal miracidia (top row of charts, red) and attenuated irradiated miracidia (bottom row of charts, grey). B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH and RNA was isolated and q-RT-PCR performed (middle chart). In the NMRI strain snail's Hsp 70 gene is repositioned after infection from an intermediate position to a more internal position within the nuclei. This repositioning is directly correlated with changes in gene expression 2 hours after infection (A). No repositioning or change in expression is observed in the BS-90 strain snails (B). No evidence for the relocation of the Hsp 70 gene loci and no induction of Hsp 70 expression were detected by qRT-PCR when the two snail lines were infected with irradiated miracidia. Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Differential Spatial Repositioning of Activated Genes in Biomphalaria glabrata Snails Infected with Schistosoma mansoni

    doi: 10.1371/journal.pntd.0003013

    Figure Lengend Snippet: Charts displaying the in vivo radial positioning and expression profile of B. glabrata Hsp 70 gene in the interphase nuclei of cells derived from NMRI (A) and BS-90 (B) snail strains, pre and post exposure to S. mansoni normal miracidia (top row of charts, red) and attenuated irradiated miracidia (bottom row of charts, grey). B. glabrata snails were infected with miracidia, dissected, fixed, and subjected to 2-D FISH and RNA was isolated and q-RT-PCR performed (middle chart). In the NMRI strain snail's Hsp 70 gene is repositioned after infection from an intermediate position to a more internal position within the nuclei. This repositioning is directly correlated with changes in gene expression 2 hours after infection (A). No repositioning or change in expression is observed in the BS-90 strain snails (B). No evidence for the relocation of the Hsp 70 gene loci and no induction of Hsp 70 expression were detected by qRT-PCR when the two snail lines were infected with irradiated miracidia. Statistically significant differences, as assessed by two-tailed Student's t-test, between normalized gene signal in each shell of control nuclei compared with infected snail nuclei are indicated by a black asterisk (P

    Article Snippet: Quantitative real time-PCR Total RNA from the whole body of B. glabrata adult snails (∼8–12 mm shell diameter) either unexposed (0 min) or exposed was extracted individually by RNAzol RT (Molecular Research Center, Inc.) according to the manufacturer's manual.

    Techniques: In Vivo, Expressing, Derivative Assay, Irradiation, Infection, Fluorescence In Situ Hybridization, Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test