geneamp rna pcr core kit  (Thermo Fisher)


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    Structured Review

    Thermo Fisher geneamp rna pcr core kit
    IL-1Ra inhibits IL-1 β , IL-6, and IL-8 mRNA expression in BRAT cells. Cells stably expressing BRAT were plated at equal densities, treated with recombinant IL-1Ra, and collected. <t>RNA</t> was isolated, DNAse-treated, and reverse-transcribed. Real-time <t>PCR</t> was performed in triplicate from two separate experiments for IL-1 β , IL-6, IL-8, and actin using SYBR Green detection. Data are expressed as the mean fold change in gene expression ± standard error.
    Geneamp Rna Pcr Core Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 73 article reviews
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    geneamp rna pcr core kit - by Bioz Stars, 2020-08
    92/100 stars

    Images

    1) Product Images from "BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells"

    Article Title: BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells

    Journal: BioMed Research International

    doi: 10.1155/2015/652017

    IL-1Ra inhibits IL-1 β , IL-6, and IL-8 mRNA expression in BRAT cells. Cells stably expressing BRAT were plated at equal densities, treated with recombinant IL-1Ra, and collected. RNA was isolated, DNAse-treated, and reverse-transcribed. Real-time PCR was performed in triplicate from two separate experiments for IL-1 β , IL-6, IL-8, and actin using SYBR Green detection. Data are expressed as the mean fold change in gene expression ± standard error.
    Figure Legend Snippet: IL-1Ra inhibits IL-1 β , IL-6, and IL-8 mRNA expression in BRAT cells. Cells stably expressing BRAT were plated at equal densities, treated with recombinant IL-1Ra, and collected. RNA was isolated, DNAse-treated, and reverse-transcribed. Real-time PCR was performed in triplicate from two separate experiments for IL-1 β , IL-6, IL-8, and actin using SYBR Green detection. Data are expressed as the mean fold change in gene expression ± standard error.

    Techniques Used: Expressing, Stable Transfection, Recombinant, Isolation, Real-time Polymerase Chain Reaction, SYBR Green Assay

    IL-1 β protein and message levels are increased in stably transfected BRAT cells. (a) Cells stably expressing BRAT or the pcDNA3.1 cells were plated at equal densities and RNA was isolated, DNAse-treated, and reverse-transcribed. Real-time PCR was performed in triplicate for IL-1 β and actin using SYBR Green detection and the data are expressed as the mean fold difference ± standard error. (b) pcDNA3.1 and BRATc1 cells were analyzed for precursor (pro-) and cleaved IL-1 β protein expression via western blot. Blots were then stripped and probed for β -actin as a loading control. Values represent relative densitometry. (c) pcDNA3.1, BRATc1, and BRATc2 cells were plated in triplicate at similar densities and conditioned media were collected. IL-1 β ELISA activity assay was performed in triplicate and the data are expressed as the mean ± standard error. Symbol ( ∗ ) denotes statistical significance at the 0.01 confidence level between BRAT and PCDNA3.1 cells.
    Figure Legend Snippet: IL-1 β protein and message levels are increased in stably transfected BRAT cells. (a) Cells stably expressing BRAT or the pcDNA3.1 cells were plated at equal densities and RNA was isolated, DNAse-treated, and reverse-transcribed. Real-time PCR was performed in triplicate for IL-1 β and actin using SYBR Green detection and the data are expressed as the mean fold difference ± standard error. (b) pcDNA3.1 and BRATc1 cells were analyzed for precursor (pro-) and cleaved IL-1 β protein expression via western blot. Blots were then stripped and probed for β -actin as a loading control. Values represent relative densitometry. (c) pcDNA3.1, BRATc1, and BRATc2 cells were plated in triplicate at similar densities and conditioned media were collected. IL-1 β ELISA activity assay was performed in triplicate and the data are expressed as the mean ± standard error. Symbol ( ∗ ) denotes statistical significance at the 0.01 confidence level between BRAT and PCDNA3.1 cells.

    Techniques Used: Stable Transfection, Transfection, Expressing, Isolation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay

    2) Product Images from "Regulatory Role of the MisR/S Two-Component System in Hemoglobin Utilization in Neisseria meningitidis ▿ ▿ †"

    Article Title: Regulatory Role of the MisR/S Two-Component System in Hemoglobin Utilization in Neisseria meningitidis ▿ ▿ †

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00363-09

    RNA isolation and real-time qRT-PCR.
    Figure Legend Snippet: RNA isolation and real-time qRT-PCR.

    Techniques Used: Isolation, Quantitative RT-PCR

    3) Product Images from "Identification of a New Splice Variant of the Human ABCC6 Transporter"

    Article Title: Identification of a New Splice Variant of the Human ABCC6 Transporter

    Journal: Research Letters in Biochemistry

    doi: 10.1155/2008/912478

    (Top) Expression pattern of exon18–exon25 region of ABCC6 from commercially available human liver and kidney cDNA (lanes 1 and 2), RNA reverse transcribed of primary human hepatocites (lane 3), and human embryonic kidney (lane 4). (Bottom) RT-PCR with β -actin primers as a control. The obtained PCR products were visualized by EtBr-stained agarose gel electrophoresis.
    Figure Legend Snippet: (Top) Expression pattern of exon18–exon25 region of ABCC6 from commercially available human liver and kidney cDNA (lanes 1 and 2), RNA reverse transcribed of primary human hepatocites (lane 3), and human embryonic kidney (lane 4). (Bottom) RT-PCR with β -actin primers as a control. The obtained PCR products were visualized by EtBr-stained agarose gel electrophoresis.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis

    4) Product Images from "Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes"

    Article Title: Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes

    Journal: Oncotarget

    doi:

    Comparative analysis of the kinetics of DNA demethylation of the APM genes induced by IFNγ or 5AC TC-1/A9 cells were cultured in the presence of either IFNγ or 5AC. For the indicated time periods, DNA samples were isolated, bisulphite treated and subjected to MSP analysis of the TAP-1, TAP-2 , LMP-2 LMP-7 promoter sequences. U = unmethylated primer, M = methylated. In untreated cells, the core CpG island was highly methylated, and demethylation was detected within 2 hours after the IFNγ treatment, while nearly complete demethylation was evident by 6 hours (A). After 5AC treatment, strong demethylation was evident by 24 hours (A). The amount of 1 μg of RNA was reverse transcribed to cDNA and the PCR products were quantified. Upregulation of APM genes in TC-1/A9 cells after the treatment with IFNγ after 2 hours (A) and with 5AC after 48 hour (B). * denote significant changes (P
    Figure Legend Snippet: Comparative analysis of the kinetics of DNA demethylation of the APM genes induced by IFNγ or 5AC TC-1/A9 cells were cultured in the presence of either IFNγ or 5AC. For the indicated time periods, DNA samples were isolated, bisulphite treated and subjected to MSP analysis of the TAP-1, TAP-2 , LMP-2 LMP-7 promoter sequences. U = unmethylated primer, M = methylated. In untreated cells, the core CpG island was highly methylated, and demethylation was detected within 2 hours after the IFNγ treatment, while nearly complete demethylation was evident by 6 hours (A). After 5AC treatment, strong demethylation was evident by 24 hours (A). The amount of 1 μg of RNA was reverse transcribed to cDNA and the PCR products were quantified. Upregulation of APM genes in TC-1/A9 cells after the treatment with IFNγ after 2 hours (A) and with 5AC after 48 hour (B). * denote significant changes (P

    Techniques Used: Cell Culture, Isolation, Methylation, Polymerase Chain Reaction

    5) Product Images from "Oncolytic adenoviral vectors which employ the survivin promoter induce glioma oncolysis via a process of beclin-dependent autophagy"

    Article Title: Oncolytic adenoviral vectors which employ the survivin promoter induce glioma oncolysis via a process of beclin-dependent autophagy

    Journal: International journal of oncology

    doi:

    Promoter dependent E1A expression in human gliomas. (A) U87MG, U118MG, U373MG, A172 and No. 10 glioma cells or (B) primary glioma slices obtained from a patient were seeded in 6-well plates overnight and then infected with CRAd-S-RGD, AdRGD or AdWT. After 4 h adsorption, cells were rinsed with PBS, unbound virus was removed and fresh growth media was added. Twenty-four or 48 h post-infection, total RNA was extracted and subjected to the quantification RT-PCR. All results are presented as E1A copy number per ng RNA. * p
    Figure Legend Snippet: Promoter dependent E1A expression in human gliomas. (A) U87MG, U118MG, U373MG, A172 and No. 10 glioma cells or (B) primary glioma slices obtained from a patient were seeded in 6-well plates overnight and then infected with CRAd-S-RGD, AdRGD or AdWT. After 4 h adsorption, cells were rinsed with PBS, unbound virus was removed and fresh growth media was added. Twenty-four or 48 h post-infection, total RNA was extracted and subjected to the quantification RT-PCR. All results are presented as E1A copy number per ng RNA. * p

    Techniques Used: Expressing, Infection, Adsorption, Reverse Transcription Polymerase Chain Reaction

    6) Product Images from "Binding and neutralization efficiencies of monoclonal antibodies, Fab fragments, and scFv specific for L1 epitopes on the capsid of infectious HPV particles"

    Article Title: Binding and neutralization efficiencies of monoclonal antibodies, Fab fragments, and scFv specific for L1 epitopes on the capsid of infectious HPV particles

    Journal: Virology

    doi: 10.1016/j.virol.2006.12.002

    Neutralization of HPV-11 assayed by nested RT-PCR. (a) For measurement of neutralization titers, A431 cells were infected with virions (150 particles/cell) which had been preincubated with 2-fold dilutions of purified MAb (H11.B2 shown as representative data). E1^E4 spliced transcripts were amplified by nested RT-PCR four days following infection. All reported titers were obtained in two independent experiments. (b) A431 cells were infected with the indicated amounts of HPV-11 particles and infection was assayed 4 days later using nested RT-PCR targeting the E1^E4 splice junction. This experiment demonstrates the high sensitivity of this infection assay as amplicons are detectable in total RNA of cells infected with fewer than 1 particle/cell.
    Figure Legend Snippet: Neutralization of HPV-11 assayed by nested RT-PCR. (a) For measurement of neutralization titers, A431 cells were infected with virions (150 particles/cell) which had been preincubated with 2-fold dilutions of purified MAb (H11.B2 shown as representative data). E1^E4 spliced transcripts were amplified by nested RT-PCR four days following infection. All reported titers were obtained in two independent experiments. (b) A431 cells were infected with the indicated amounts of HPV-11 particles and infection was assayed 4 days later using nested RT-PCR targeting the E1^E4 splice junction. This experiment demonstrates the high sensitivity of this infection assay as amplicons are detectable in total RNA of cells infected with fewer than 1 particle/cell.

    Techniques Used: Neutralization, Reverse Transcription Polymerase Chain Reaction, Infection, Purification, Amplification

    Neutralization of HPV-11 virions by scFv of H11.B2. (a) HPV-11 virions were preincubated with H11.B2 Fab fragments or with the H11.B2 scFv (scB2) prior to infecting A431 cells (150 particles/cell). Four days post-infection, total RNA was assayed for the presence of E1^E4 transcripts using QRT-PCR. (b) Half-maximal binding concentrations were determined by ELISA using HPV-11 L1-only VLPs as antigen. HPV-11 neutralization titers (as EC 50 .
    Figure Legend Snippet: Neutralization of HPV-11 virions by scFv of H11.B2. (a) HPV-11 virions were preincubated with H11.B2 Fab fragments or with the H11.B2 scFv (scB2) prior to infecting A431 cells (150 particles/cell). Four days post-infection, total RNA was assayed for the presence of E1^E4 transcripts using QRT-PCR. (b) Half-maximal binding concentrations were determined by ELISA using HPV-11 L1-only VLPs as antigen. HPV-11 neutralization titers (as EC 50 .

    Techniques Used: Neutralization, Infection, Quantitative RT-PCR, Binding Assay, Enzyme-linked Immunosorbent Assay

    Neutralization of HPV-11 virions by secreted scFv. (a) HPV-11 virions were incubated with various High 5 supernatants prior to infection of A431 cells. Virions were incubated with spent medium of either uninfected cells (Mock) or cells infected with recombinant baculoviruses containing genes for secreted GST-scFv fusion proteins. E1^E4 transcripts in total RNA were assayed 4 days post-infection using nested RT-PCR. Similar results were seen with supernatants of infected Sf9 cells (not shown). (b) High 5 cell supernatants containing secreted GST-scFv fusion proteins were preincubated with HPV-11 virions prior to infection of A431 cells. Infection was assayed 4 days post-infection by QRT-PCR. GST-B1.A1 is a non-functional scFv for the BPV-1 specific antibody B1.A1 and was used here as a negative control.
    Figure Legend Snippet: Neutralization of HPV-11 virions by secreted scFv. (a) HPV-11 virions were incubated with various High 5 supernatants prior to infection of A431 cells. Virions were incubated with spent medium of either uninfected cells (Mock) or cells infected with recombinant baculoviruses containing genes for secreted GST-scFv fusion proteins. E1^E4 transcripts in total RNA were assayed 4 days post-infection using nested RT-PCR. Similar results were seen with supernatants of infected Sf9 cells (not shown). (b) High 5 cell supernatants containing secreted GST-scFv fusion proteins were preincubated with HPV-11 virions prior to infection of A431 cells. Infection was assayed 4 days post-infection by QRT-PCR. GST-B1.A1 is a non-functional scFv for the BPV-1 specific antibody B1.A1 and was used here as a negative control.

    Techniques Used: Neutralization, Incubation, Infection, Recombinant, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Functional Assay, Negative Control

    7) Product Images from "Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling"

    Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/s13058-014-0479-6

    Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (** P
    Figure Legend Snippet: Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (** P

    Techniques Used: Recombinant, Incubation, Western Blot, shRNA, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Extracellular matrix protein 1 induces matrix metalloproteinase 9 transcription. (A) At 24 hours after seeding, cells were incubated with serum-free medium for a further 24 hours. Supernatant medium from each cell line was reacted with matrix metalloproteinase 9 (MMP9) substrate, and relative fluorescence units were determined at 480 to 620 nm. Cont, Control; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) MMP9 mRNA levels were determined by real-time PCR using primers specific for MMP9 (* P
    Figure Legend Snippet: Extracellular matrix protein 1 induces matrix metalloproteinase 9 transcription. (A) At 24 hours after seeding, cells were incubated with serum-free medium for a further 24 hours. Supernatant medium from each cell line was reacted with matrix metalloproteinase 9 (MMP9) substrate, and relative fluorescence units were determined at 480 to 620 nm. Cont, Control; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) MMP9 mRNA levels were determined by real-time PCR using primers specific for MMP9 (* P

    Techniques Used: Incubation, Fluorescence, shRNA, Plasmid Preparation, Real-time Polymerase Chain Reaction

    8) Product Images from "Treatment of endometriosis with a VEGF targeted conditionally replicative adenovirus"

    Article Title: Treatment of endometriosis with a VEGF targeted conditionally replicative adenovirus

    Journal: Fertility and sterility

    doi: 10.1016/j.fertnstert.2009.04.042

    (A) The human VEGF gene is overexpressed in ectopic endometriosis. RNA was extracted from deep infiltrating endometriotic lesions, eutopic endometrium and normal peritoneum from four patients suffering from deep infiltrating endometriosis and reverse-transcribed into cDNA. Real-time PCR analysis was performed to quantify the expression of the VEGF gene. The gene copy numbers are normalized by the GAPDH copy number. Error bars indicate SD. *p
    Figure Legend Snippet: (A) The human VEGF gene is overexpressed in ectopic endometriosis. RNA was extracted from deep infiltrating endometriotic lesions, eutopic endometrium and normal peritoneum from four patients suffering from deep infiltrating endometriosis and reverse-transcribed into cDNA. Real-time PCR analysis was performed to quantify the expression of the VEGF gene. The gene copy numbers are normalized by the GAPDH copy number. Error bars indicate SD. *p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    9) Product Images from "The Effects of Methionine Acquisition and Synthesis on Streptococcus Pneumoniae Growth and Virulence"

    Article Title: The Effects of Methionine Acquisition and Synthesis on Streptococcus Pneumoniae Growth and Virulence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049638

    Gene expression of Sp_0149 during infection. ( A ) Ethidium stained agarose gel showing equal amplification efficiency for the primer pairs used for PCR of psaA and metQ when using S. pneumoniae 0100993 genomic DNA as the template. ( B ) Ethidium stained agarose gel of cDNA products generated by RT-PCR after 26 and 30 cycles using S. pneumoniae 0100993 total RNA extracted from the blood of infected mice and primers for 16S rRNA (internal control), psaA (positive control) and metQ . (C) Relative mRNA concentrations of selected genes of S. pneumoniae WCH43 (serotype 4) in various in vivo niches at 72 h post-intranasal infection of mice. Transcript abundance for each gene was obtained by microarray analysis, and normalized against those obtained for the 16S rRNA control. Quantitative fold differences for each transcript were determined as a ratio of its abundance to that of the 16S rRNA control. Data are means ± SEM for each gene transcript from three replicate challenge experiments.
    Figure Legend Snippet: Gene expression of Sp_0149 during infection. ( A ) Ethidium stained agarose gel showing equal amplification efficiency for the primer pairs used for PCR of psaA and metQ when using S. pneumoniae 0100993 genomic DNA as the template. ( B ) Ethidium stained agarose gel of cDNA products generated by RT-PCR after 26 and 30 cycles using S. pneumoniae 0100993 total RNA extracted from the blood of infected mice and primers for 16S rRNA (internal control), psaA (positive control) and metQ . (C) Relative mRNA concentrations of selected genes of S. pneumoniae WCH43 (serotype 4) in various in vivo niches at 72 h post-intranasal infection of mice. Transcript abundance for each gene was obtained by microarray analysis, and normalized against those obtained for the 16S rRNA control. Quantitative fold differences for each transcript were determined as a ratio of its abundance to that of the 16S rRNA control. Data are means ± SEM for each gene transcript from three replicate challenge experiments.

    Techniques Used: Expressing, Infection, Staining, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Generated, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Positive Control, In Vivo, Microarray

    10) Product Images from "Interaction of Staufen1 with the 5? end of mRNA facilitates translation of these RNAs"

    Article Title: Interaction of Staufen1 with the 5? end of mRNA facilitates translation of these RNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki794

    Binding of Stau1 55 to the 5′ end increases translation of structure-repressed transcripts. (A) Schematic representation of 5′-structure-repressed transcripts. RNAs coding for the R luc reporter protein are shown with one copy of the SBS or two copies of the MS2-binding site (MS2bs) at the 5′ end. (B) HEK293T cells were co-transfected with plasmids expressing either R luc or SBS- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of Stau1 55 -HA 3 , a 100-fold repression of translation of the SBS- R luc RNA was observed as compared with translation of R luc RNA. Results are expressed as luciferase activity versus concentration of the Stau1 55 -HA 3 coding plasmid. To facilitate comparison, the luciferase activity in the absence of Stau1 55 -HA 3 was defined as 1. P ≤ 0.01, n = 3. Black bars, SBS- R luc RNA; hatched bars, R luc RNA. (C) HEK293T cells were co-transfected with plasmids expressing the SBS- R luc transcript and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (D) HEK293T cells were co-transfected with plasmids expressing the MS2bs- R luc transcript and different concentrations of plasmids coding for either MS2-Stau1 55 -HA 3 , MS2-HA or Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of MS2-Stau1 55 -HA 3 , a 100-fold repression of translation of the MS2bs- R luc RNA was observed as compared with translation of R luc RNA. To facilitate comparison, the luciferase activity in the absence of expressor plasmids was defined as 1, n = 3.
    Figure Legend Snippet: Binding of Stau1 55 to the 5′ end increases translation of structure-repressed transcripts. (A) Schematic representation of 5′-structure-repressed transcripts. RNAs coding for the R luc reporter protein are shown with one copy of the SBS or two copies of the MS2-binding site (MS2bs) at the 5′ end. (B) HEK293T cells were co-transfected with plasmids expressing either R luc or SBS- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of Stau1 55 -HA 3 , a 100-fold repression of translation of the SBS- R luc RNA was observed as compared with translation of R luc RNA. Results are expressed as luciferase activity versus concentration of the Stau1 55 -HA 3 coding plasmid. To facilitate comparison, the luciferase activity in the absence of Stau1 55 -HA 3 was defined as 1. P ≤ 0.01, n = 3. Black bars, SBS- R luc RNA; hatched bars, R luc RNA. (C) HEK293T cells were co-transfected with plasmids expressing the SBS- R luc transcript and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (D) HEK293T cells were co-transfected with plasmids expressing the MS2bs- R luc transcript and different concentrations of plasmids coding for either MS2-Stau1 55 -HA 3 , MS2-HA or Stau1 55 -HA 3 . Resulting luciferase activity was quantified 24 h post-transfection. In the absence of MS2-Stau1 55 -HA 3 , a 100-fold repression of translation of the MS2bs- R luc RNA was observed as compared with translation of R luc RNA. To facilitate comparison, the luciferase activity in the absence of expressor plasmids was defined as 1, n = 3.

    Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Concentration Assay, Isolation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    Stau1 55 mediated translational up-regulation does not involved RNA modification. (A) TAR-CAT RNA was incubated in RRL in the presence of 400 nM of bacterially expressed and purified Stau1 55 Δ2-his 6 or BSA for increasing periods of time. TAR-CAT RNA was then reverse transcribed and PCR amplified for 14 cycles to stay in the non-saturated part of the amplification curve. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (right panel). (B) HEK293T cells were co-transfected with plasmids expressing either R luc or TAR- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (C) Bacterially expressed and column-purified Stau1 55 Δ2-his 6 (Stau) and La-his 6 (La) (left panel) were incubated with [ 32 P]labelled double-stranded RNA in the presence of different combinations of ribonucleotides (right panel). RNA was resolved on agarose gel and revealed by autoradiography. While La-his 6 displayed an helicase activity, Stau1 55 Δ2-his 6 was inactive in this assay.
    Figure Legend Snippet: Stau1 55 mediated translational up-regulation does not involved RNA modification. (A) TAR-CAT RNA was incubated in RRL in the presence of 400 nM of bacterially expressed and purified Stau1 55 Δ2-his 6 or BSA for increasing periods of time. TAR-CAT RNA was then reverse transcribed and PCR amplified for 14 cycles to stay in the non-saturated part of the amplification curve. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (right panel). (B) HEK293T cells were co-transfected with plasmids expressing either R luc or TAR- R luc transcripts and different concentrations of a plasmid coding for Stau1 55 -HA 3 . Twenty-four hours post-transfection, RNA was isolated, reverse transcribed and PCR amplified. Resulting DNA was resolved on agarose gel. As control, the same experiment was performed in the absence of reverse transcriptase (−RT). RNA coding for GAPDH was RT–PCR and used to normalize the results. (C) Bacterially expressed and column-purified Stau1 55 Δ2-his 6 (Stau) and La-his 6 (La) (left panel) were incubated with [ 32 P]labelled double-stranded RNA in the presence of different combinations of ribonucleotides (right panel). RNA was resolved on agarose gel and revealed by autoradiography. While La-his 6 displayed an helicase activity, Stau1 55 Δ2-his 6 was inactive in this assay.

    Techniques Used: Modification, Incubation, Purification, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Transfection, Expressing, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction, Autoradiography, Activity Assay

    11) Product Images from "15-Lipoxygenase-1 Production is Lost in Pancreatic Cancer and Overexpression of the Gene Inhibits Tumor Cell Growth 1"

    Article Title: 15-Lipoxygenase-1 Production is Lost in Pancreatic Cancer and Overexpression of the Gene Inhibits Tumor Cell Growth 1

    Journal:

    doi:

    Lack of 15-LOX-1 mRNA expression in pancreatic cancer cell lines compared to human monocytes (positive control). Total RNA was isolated, reverse-transcribed, and then amplified by PCR. The PCR product was separated on 1% agarose gel and visualized by
    Figure Legend Snippet: Lack of 15-LOX-1 mRNA expression in pancreatic cancer cell lines compared to human monocytes (positive control). Total RNA was isolated, reverse-transcribed, and then amplified by PCR. The PCR product was separated on 1% agarose gel and visualized by

    Techniques Used: Expressing, Positive Control, Isolation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    12) Product Images from "Activation of Liver X Receptor Inhibits Osteopontin and Ameliorates Diabetic Nephropathy"

    Article Title: Activation of Liver X Receptor Inhibits Osteopontin and Ameliorates Diabetic Nephropathy

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2012010022

    LXR agonist suppresses diabetes-induced OPN mRNA and protein expressions in the kidney. (A) Total RNA was isolated from the kidney samples and analyzed for the OPN mRNA expression levels by qRT-PCR. Renal OPN mRNA expression is significantly increased
    Figure Legend Snippet: LXR agonist suppresses diabetes-induced OPN mRNA and protein expressions in the kidney. (A) Total RNA was isolated from the kidney samples and analyzed for the OPN mRNA expression levels by qRT-PCR. Renal OPN mRNA expression is significantly increased

    Techniques Used: Isolation, Expressing, Quantitative RT-PCR

    13) Product Images from "Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes"

    Article Title: Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes

    Journal: Virology Journal

    doi: 10.1186/1743-422X-4-115

    Viral genetic stability and artifactual DNA amplification of the EGFP gene. (A) Agarose gel electrophoresis of plasmid T3 DNA without and with the EGFP cassete (lanes 1 and 2, respectively); DNA amplification of plasmid T3 and the recombinant one (lanes 3 and 4, respectively); RT-PCR on RNA of YF17D/E200T3 and YF17D/Esa/5.1glic 2P viruses without and with the EGFP cassete (lanes 5 and 6, respectively). (B) Schematic representation of the amplification based on the correct annealing of the E protein gene (black bars) and the EGFP stem-anchor (white bars) domains from two different DNA strands yielding an amplicon of 2,030 bp. (C) and (D) schematic representation of the amplification based on the spurious alternative annealing possibilities of the E protein gene (black bars) and the EGFP stem-anchor (white bars) regions from two different DNA strands yielding amplicons of 1,001 bp (without the EGFP cassete and with a single stem-anchor domain, gray bars) or 3,059 bp (with the duplicated EGFP gene and an extra copy of stem-anchor region), respectively.
    Figure Legend Snippet: Viral genetic stability and artifactual DNA amplification of the EGFP gene. (A) Agarose gel electrophoresis of plasmid T3 DNA without and with the EGFP cassete (lanes 1 and 2, respectively); DNA amplification of plasmid T3 and the recombinant one (lanes 3 and 4, respectively); RT-PCR on RNA of YF17D/E200T3 and YF17D/Esa/5.1glic 2P viruses without and with the EGFP cassete (lanes 5 and 6, respectively). (B) Schematic representation of the amplification based on the correct annealing of the E protein gene (black bars) and the EGFP stem-anchor (white bars) domains from two different DNA strands yielding an amplicon of 2,030 bp. (C) and (D) schematic representation of the amplification based on the spurious alternative annealing possibilities of the E protein gene (black bars) and the EGFP stem-anchor (white bars) regions from two different DNA strands yielding amplicons of 1,001 bp (without the EGFP cassete and with a single stem-anchor domain, gray bars) or 3,059 bp (with the duplicated EGFP gene and an extra copy of stem-anchor region), respectively.

    Techniques Used: Amplification, Agarose Gel Electrophoresis, Plasmid Preparation, Recombinant, Reverse Transcription Polymerase Chain Reaction

    Molecular cloning of EGFP protein expression cassete in the chimeric YF17D/DEN4 virus genome. (A) Schematic representation of YF 17D/DEN4/Esa/EGFP/6 recombinant virus genome and the genetic elements fused to EGFP gene. (B) Growth of recombinant YF17D/DEN4 viruses in Vero cells. Three independent experiments were performed to measure viral spread in Vero cells after infection with an multiplicity of infection (MOI) of 0.02. Cell culture supernatant aliquots were taken at 24, 48, 72, 96, 120 and 140 hour post-infection (p.i.) and titrated by plaque formation on Vero cell monolayers. (C) Analysis of recombinant YF 17D/DEN4/Esa/6 virus genetic stability after serial passaging on Vero cell monolayers. Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted from samples of the supernatant of cultures according to the passage numbering indicated on top of each lane. The first lane corresponds to cDNA-derived YF17D/DEN4 virus RNA; the remaining lanes are RT-PCR profiles from YF17D/DEN4/Esa/6 virus RNA at different passage levels with lanes 2 and 3 corresponding to amplicons from RNAs of viral stocks (1P, transfection supernatant) or passage two (2P, first passage of transfection supernatant), respectively. Lanes 4 to 11 represent RT-PCR products, which were obtained from viral RNA in the fifth, tenth, 15 th and 20 th passages of the two independent passage lineages (5P1 and 5P2; 10P1 and 10P2, 15P1 and 15P2, 20P1 and 20P2, respectively).
    Figure Legend Snippet: Molecular cloning of EGFP protein expression cassete in the chimeric YF17D/DEN4 virus genome. (A) Schematic representation of YF 17D/DEN4/Esa/EGFP/6 recombinant virus genome and the genetic elements fused to EGFP gene. (B) Growth of recombinant YF17D/DEN4 viruses in Vero cells. Three independent experiments were performed to measure viral spread in Vero cells after infection with an multiplicity of infection (MOI) of 0.02. Cell culture supernatant aliquots were taken at 24, 48, 72, 96, 120 and 140 hour post-infection (p.i.) and titrated by plaque formation on Vero cell monolayers. (C) Analysis of recombinant YF 17D/DEN4/Esa/6 virus genetic stability after serial passaging on Vero cell monolayers. Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted from samples of the supernatant of cultures according to the passage numbering indicated on top of each lane. The first lane corresponds to cDNA-derived YF17D/DEN4 virus RNA; the remaining lanes are RT-PCR profiles from YF17D/DEN4/Esa/6 virus RNA at different passage levels with lanes 2 and 3 corresponding to amplicons from RNAs of viral stocks (1P, transfection supernatant) or passage two (2P, first passage of transfection supernatant), respectively. Lanes 4 to 11 represent RT-PCR products, which were obtained from viral RNA in the fifth, tenth, 15 th and 20 th passages of the two independent passage lineages (5P1 and 5P2; 10P1 and 10P2, 15P1 and 15P2, 20P1 and 20P2, respectively).

    Techniques Used: Molecular Cloning, Expressing, Recombinant, Infection, Cell Culture, Passaging, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Transfection

    Analysis of recombinant virus genetic stability after serial passaging. (A) Schematics of viral regeneration and subsequent passages (10) of the YF 17D/Esa/5.1 glic virus obtained after RNA transfection. Two independent series of serial passages (at MOI of 0.02); P1 and P2 were analyzed by RT-PCR and flow citometry at passages 5 and 10 and are represented in all panels as 5P1, 10P1, 5P2 and 10P2. In these experiments the YF17D/E200-T3 virus was used as negative control for EGFP expression. (B) Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted of samples from the supernatant of cultures used to derive the citometry data (C) according the passage history (A). The length of the main RT-PCR bands are shown on the left side. (C) The rate of double gated cells (YF+, EGFP+) over the total YF+ gated cells (YF+, EGFP+ plus YF+, EGFP- gated cells) corresponds to the percentage of cells infected by YF 17D/Esa/5.1 glic virus stably expressing the EGFP protein. The respective columns indicate the values for each of the viral passages.
    Figure Legend Snippet: Analysis of recombinant virus genetic stability after serial passaging. (A) Schematics of viral regeneration and subsequent passages (10) of the YF 17D/Esa/5.1 glic virus obtained after RNA transfection. Two independent series of serial passages (at MOI of 0.02); P1 and P2 were analyzed by RT-PCR and flow citometry at passages 5 and 10 and are represented in all panels as 5P1, 10P1, 5P2 and 10P2. In these experiments the YF17D/E200-T3 virus was used as negative control for EGFP expression. (B) Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted of samples from the supernatant of cultures used to derive the citometry data (C) according the passage history (A). The length of the main RT-PCR bands are shown on the left side. (C) The rate of double gated cells (YF+, EGFP+) over the total YF+ gated cells (YF+, EGFP+ plus YF+, EGFP- gated cells) corresponds to the percentage of cells infected by YF 17D/Esa/5.1 glic virus stably expressing the EGFP protein. The respective columns indicate the values for each of the viral passages.

    Techniques Used: Recombinant, Passaging, Transfection, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Negative Control, Expressing, Infection, Stable Transfection

    14) Product Images from "AXL is an oncotarget in human colorectal cancer"

    Article Title: AXL is an oncotarget in human colorectal cancer

    Journal: Oncotarget

    doi:

    Expression and activation of AXL in human CRC cell lines A. 300 μg of protein lysates were obtained from human CRC cell lines SW620, SW480, LOVO, HCT116 and were analysed by human phospho-kinase array evaluating the following receptors: AXL, EphA1, EphA2, EphA3, EphA6, EphA4, EphA7, EphB1 EphB2, EphB4, EphB6, ErbB2, ErbB3, ErbB4, EGFR, FGF R1, FGFR2a, FGF R3, FGFR4, Flt 3, HGF R, insulinR, IGF-I R, Mer, MSPR, MCSFR, MuSK, PDGFrα, PDGrβ, SCFR, cRET, ROR1, ROR2, Tie2, TrkA, TrkB, TrkC, VEGFR1, VEGFR2, VEGFR3, Tie1. B. Western blot analysis of AXL receptor in SW48, SW48-CR, HT29, LOVO, SW620, HCT15, HCT116, GEO, GEO-CR, COLO205, SW480. Thirty micrograms of cell protein extracts were fractionated through 4% to 20% SDS-PAGE, transferred to nitrocellulose filters, and incubated with the indicated antibodies as described in Materials and Methods. Immunoreactive proteins were visualized by enhanced chemiluminescence. C. specific mRNA expression by quantitative real time-PCR: total RNA was extracted from colorectal (SW620, SW48, SW480, HT29, LOVO, HCT15, HCT116) and thyroid cancer cell lines (TPC, CAL 62) quantitative real time-PCR was done to assess the expression of AXL and GAS6 mRNA. D. GAS6 protein levels were measured in cell culture media of CRC cancer (SW620, SW48, SW480, HT29, LOVO, HCT15, HCT116) and thyroid (TPC, NIM) cancer cell lines by using specific ELISA as described in Materials and Methods.
    Figure Legend Snippet: Expression and activation of AXL in human CRC cell lines A. 300 μg of protein lysates were obtained from human CRC cell lines SW620, SW480, LOVO, HCT116 and were analysed by human phospho-kinase array evaluating the following receptors: AXL, EphA1, EphA2, EphA3, EphA6, EphA4, EphA7, EphB1 EphB2, EphB4, EphB6, ErbB2, ErbB3, ErbB4, EGFR, FGF R1, FGFR2a, FGF R3, FGFR4, Flt 3, HGF R, insulinR, IGF-I R, Mer, MSPR, MCSFR, MuSK, PDGFrα, PDGrβ, SCFR, cRET, ROR1, ROR2, Tie2, TrkA, TrkB, TrkC, VEGFR1, VEGFR2, VEGFR3, Tie1. B. Western blot analysis of AXL receptor in SW48, SW48-CR, HT29, LOVO, SW620, HCT15, HCT116, GEO, GEO-CR, COLO205, SW480. Thirty micrograms of cell protein extracts were fractionated through 4% to 20% SDS-PAGE, transferred to nitrocellulose filters, and incubated with the indicated antibodies as described in Materials and Methods. Immunoreactive proteins were visualized by enhanced chemiluminescence. C. specific mRNA expression by quantitative real time-PCR: total RNA was extracted from colorectal (SW620, SW48, SW480, HT29, LOVO, HCT15, HCT116) and thyroid cancer cell lines (TPC, CAL 62) quantitative real time-PCR was done to assess the expression of AXL and GAS6 mRNA. D. GAS6 protein levels were measured in cell culture media of CRC cancer (SW620, SW48, SW480, HT29, LOVO, HCT15, HCT116) and thyroid (TPC, NIM) cancer cell lines by using specific ELISA as described in Materials and Methods.

    Techniques Used: Expressing, Activation Assay, Western Blot, SDS Page, Incubation, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    15) Product Images from "Induction of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced anti-HIV-1 activity"

    Article Title: Induction of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced anti-HIV-1 activity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20051512

    IFN-α augments induction of APOBEC3 family members. (A) RNA was isolated from macrophages (two donors shown) untreated or treated with 10 ng/ml IFN-α for 4 h and evaluated by RT-PCR with primers corresponding to each of the indicated APOBEC3 genes and PKR (30 cycles). GAPDH was used as a parallel control. (B) Monocytes were transfected with APOBEC3G siRNA, cultured for 6 d, stimulated with IFN-α for 4 h, and APOBEC3G and PKR expression was monitored by RT-PCR in comparison with the GAPDH control. (C) Western blot for APOBEC3G protein 6 d after monocyte transfection with siRNA or control RNA, or electroporation only control (EPC), and treatment with or without IFN-α for 20 h. Representative experiment, n = 3.
    Figure Legend Snippet: IFN-α augments induction of APOBEC3 family members. (A) RNA was isolated from macrophages (two donors shown) untreated or treated with 10 ng/ml IFN-α for 4 h and evaluated by RT-PCR with primers corresponding to each of the indicated APOBEC3 genes and PKR (30 cycles). GAPDH was used as a parallel control. (B) Monocytes were transfected with APOBEC3G siRNA, cultured for 6 d, stimulated with IFN-α for 4 h, and APOBEC3G and PKR expression was monitored by RT-PCR in comparison with the GAPDH control. (C) Western blot for APOBEC3G protein 6 d after monocyte transfection with siRNA or control RNA, or electroporation only control (EPC), and treatment with or without IFN-α for 20 h. Representative experiment, n = 3.

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Transfection, Cell Culture, Expressing, Western Blot, Electroporation

    IFN-α induces APOBEC3G expression. (A) Macrophages were not treated (control) or treated with 1–100 ng/ml IFN-α for 18 h in the absence or presence of 1 mM 2-AP, and RNA was analyzed for APOBEC3G by RT-PCR (30 cycles). (B) Macrophages were treated or not with 10 ng/ml IFN-α for the indicated times, and RNA was subjected to RT-PCR to amplify APOBEC3G and PKR mRNA. Representative experiment, n = 3. (C) Macrophages were treated with IFN-α for 2–16 h, and APOBEC3G expression was assessed by real-time PCR as compared with untreated cells cultured in parallel. Data (mean ± SD of triplicates) are presented as fold change from 2-h control macrophages. (D) Cell lysates were obtained after macrophage treatment with 10 ng/ml IFN-α and/or 1 mM 2-AP and assessed for APOBEC3G protein (∼42 kD) by Western blot.
    Figure Legend Snippet: IFN-α induces APOBEC3G expression. (A) Macrophages were not treated (control) or treated with 1–100 ng/ml IFN-α for 18 h in the absence or presence of 1 mM 2-AP, and RNA was analyzed for APOBEC3G by RT-PCR (30 cycles). (B) Macrophages were treated or not with 10 ng/ml IFN-α for the indicated times, and RNA was subjected to RT-PCR to amplify APOBEC3G and PKR mRNA. Representative experiment, n = 3. (C) Macrophages were treated with IFN-α for 2–16 h, and APOBEC3G expression was assessed by real-time PCR as compared with untreated cells cultured in parallel. Data (mean ± SD of triplicates) are presented as fold change from 2-h control macrophages. (D) Cell lysates were obtained after macrophage treatment with 10 ng/ml IFN-α and/or 1 mM 2-AP and assessed for APOBEC3G protein (∼42 kD) by Western blot.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Cell Culture, Western Blot

    16) Product Images from "Rsu1-dependent control of PTEN expression is regulated via ATF2 and cJun"

    Article Title: Rsu1-dependent control of PTEN expression is regulated via ATF2 and cJun

    Journal: Journal of Cell Communication and Signaling

    doi: 10.1007/s12079-018-00504-4

    Rsu1-dependent changes in AKT signaling pathway. a MCF10A, T47D and MDA-MB-231 cells were infected with adenoviral vector encoding HA-tagged Rsu1 or empty vector. At 72 h post infection cell lysates were prepared and analyzed by western blot with phosphorylation specific antibodies, phospho-AKT (T308) and phospho-AKT (S473). Antibodies directed against total AKT, ILK, Rac1 and Rsu1were used to determine expression levels of those proteins. Quantitation shows the level of phosphorylated AKT normalized to total AKT and the level set in the control lane = 1. b MCF10A lysates were isolated from control siRNA and Rsu1 siRNA transfected MCF10A cells after EGF treatment for the indicated times. Immunoblot analysis was performed with equal amounts of protein samples and separated by SDS-PAGE for the levels of phospho-MKK4 (S80), phospho-AKT (p S473), total MKK4, AKT and PTEN. α-tubulin and Rsu1 antibodies were used for internal and depletion controls. c MCF10A RNA from control siRNA and Rsu1 siRNA transfected cells was used to determination level of PTEN RNA by quantitative real-time PCR. Quantitation is expressed as fold stimulation or relative amount compared to T = 0 for control siRNA transfected cells set as 1
    Figure Legend Snippet: Rsu1-dependent changes in AKT signaling pathway. a MCF10A, T47D and MDA-MB-231 cells were infected with adenoviral vector encoding HA-tagged Rsu1 or empty vector. At 72 h post infection cell lysates were prepared and analyzed by western blot with phosphorylation specific antibodies, phospho-AKT (T308) and phospho-AKT (S473). Antibodies directed against total AKT, ILK, Rac1 and Rsu1were used to determine expression levels of those proteins. Quantitation shows the level of phosphorylated AKT normalized to total AKT and the level set in the control lane = 1. b MCF10A lysates were isolated from control siRNA and Rsu1 siRNA transfected MCF10A cells after EGF treatment for the indicated times. Immunoblot analysis was performed with equal amounts of protein samples and separated by SDS-PAGE for the levels of phospho-MKK4 (S80), phospho-AKT (p S473), total MKK4, AKT and PTEN. α-tubulin and Rsu1 antibodies were used for internal and depletion controls. c MCF10A RNA from control siRNA and Rsu1 siRNA transfected cells was used to determination level of PTEN RNA by quantitative real-time PCR. Quantitation is expressed as fold stimulation or relative amount compared to T = 0 for control siRNA transfected cells set as 1

    Techniques Used: Multiple Displacement Amplification, Infection, Plasmid Preparation, Western Blot, Expressing, Quantitation Assay, Isolation, Transfection, SDS Page, Real-time Polymerase Chain Reaction

    PTEN gene regulation by MKK4-p38 Mapk signaling and Rsu1-dependent expression of PTEN in MCF10A cells. a Determination of PTEN RNA levels was measured by quantitative real-time PCR following transfection with siRNAs for the following: Rsu1, p38α, MKK3, MKK4 and MKK6. The 18S RNA was used for normalization. Bar graphs show the fold change of PTEN expression quantified as the ratios to the control siRNA level. The data show means ± SD, n = 3. b RNA levels of PTEN were measured by quantitative real-time PCR following SB 203580 treatment of MCF10A cells (0, 5 or 20 μM). The 18S RNA was used for normalization. Bar graphs show the fold change of PTEN expression quantified as the ratios to the control, the means ± SD, n = 3. c Immunoblot analysis was performed with equal amounts of protein samples for the levels of phospho-AKT (S473) and phospho-p38 (T180/Y182), total AKT, p38, PTEN expression and α-tubulin was for internal controls
    Figure Legend Snippet: PTEN gene regulation by MKK4-p38 Mapk signaling and Rsu1-dependent expression of PTEN in MCF10A cells. a Determination of PTEN RNA levels was measured by quantitative real-time PCR following transfection with siRNAs for the following: Rsu1, p38α, MKK3, MKK4 and MKK6. The 18S RNA was used for normalization. Bar graphs show the fold change of PTEN expression quantified as the ratios to the control siRNA level. The data show means ± SD, n = 3. b RNA levels of PTEN were measured by quantitative real-time PCR following SB 203580 treatment of MCF10A cells (0, 5 or 20 μM). The 18S RNA was used for normalization. Bar graphs show the fold change of PTEN expression quantified as the ratios to the control, the means ± SD, n = 3. c Immunoblot analysis was performed with equal amounts of protein samples for the levels of phospho-AKT (S473) and phospho-p38 (T180/Y182), total AKT, p38, PTEN expression and α-tubulin was for internal controls

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection

    17) Product Images from "Mutations in LRRK2 impair NF-κB pathway in iPSC-derived neurons"

    Article Title: Mutations in LRRK2 impair NF-κB pathway in iPSC-derived neurons

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0761-x

    Effect of endogenous LRRK2 silencing on NF-κB target genes. a-h qRT-PCR analyses of inflammatory genes 5 days after LRRK2 silencing. The effect of LRRK2 silencing was evident in the regulation of RNA levels of COX-2 , A20 , and TNFR1 . For COX-2 , the effect of LRRK2 silencing was significant (two-way ANOVA, ** P = 0.0021,), and post hoc analyses revealed a significant effect of shLRRK2 on RNA levels specifically in LRRK2 G2019S neurons (** P
    Figure Legend Snippet: Effect of endogenous LRRK2 silencing on NF-κB target genes. a-h qRT-PCR analyses of inflammatory genes 5 days after LRRK2 silencing. The effect of LRRK2 silencing was evident in the regulation of RNA levels of COX-2 , A20 , and TNFR1 . For COX-2 , the effect of LRRK2 silencing was significant (two-way ANOVA, ** P = 0.0021,), and post hoc analyses revealed a significant effect of shLRRK2 on RNA levels specifically in LRRK2 G2019S neurons (** P

    Techniques Used: Quantitative RT-PCR

    18) Product Images from "Phorbol 12-Myristate 13-Acetate Induces MUC16 Expression via PKC? and p38 in Human Airway Epithelial Cells"

    Article Title: Phorbol 12-Myristate 13-Acetate Induces MUC16 Expression via PKC? and p38 in Human Airway Epithelial Cells

    Journal: Clinical and Experimental Otorhinolaryngology

    doi: 10.3342/ceo.2012.5.3.161

    The effect of p38 MAPK siRNA on the phosphorylation of p38 MAPK in PMA-induced MUC16 mRNA expression in the NCI-H292 airway epithelial cells. MUC16 mRNA expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR. The knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. * P
    Figure Legend Snippet: The effect of p38 MAPK siRNA on the phosphorylation of p38 MAPK in PMA-induced MUC16 mRNA expression in the NCI-H292 airway epithelial cells. MUC16 mRNA expression was analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR. The knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC16 mRNA expression. MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. * P

    Techniques Used: Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Small Interfering RNA

    19) Product Images from "Functional analysis of the promoter of the mitochondrial phosphate carrier human gene: identification of activator and repressor elements and their transcription factors"

    Article Title: Functional analysis of the promoter of the mitochondrial phosphate carrier human gene: identification of activator and repressor elements and their transcription factors

    Journal: Biochemical Journal

    doi: 10.1042/BJ20050776

    Effect of forskolin on PiC promoter activity and on PiC content both at the transcript and protein levels ( A ) Effect of forskolin on PiC promoter activity conferred by the DNA fragment −223/+95 with or without mutation in the CREB binding site. Transfected HeLa cells treated or untreated with 10 μg/ml forskolin were assayed for CAT expression activity. Grey bars indicate HeLa cells transfected with the wild-type (W) DNA fragment −223/+95 and the DNA fragment mutated in the CREB site (M). The black bars indicate forskolin-treated HeLa cells transfected with the constructs W and M. Means±S.D. for three duplicate independent experiments are shown. ( B ) Effect of forskolin on the PiC mRNA level. RT–PCR was performed with specific primers for the cDNA of PiC or β-actin and with total RNA from HeLa cells treated or untreated with forskolin. ( C ) Effect of forskolin on the PiC protein content. The PiC protein of HeLa cells treated or untreated with forskolin was immunodecorated with an anti-N-terminal PiC antibody. The reactivity of the antibody to recombinant human PiC-B was also tested.
    Figure Legend Snippet: Effect of forskolin on PiC promoter activity and on PiC content both at the transcript and protein levels ( A ) Effect of forskolin on PiC promoter activity conferred by the DNA fragment −223/+95 with or without mutation in the CREB binding site. Transfected HeLa cells treated or untreated with 10 μg/ml forskolin were assayed for CAT expression activity. Grey bars indicate HeLa cells transfected with the wild-type (W) DNA fragment −223/+95 and the DNA fragment mutated in the CREB site (M). The black bars indicate forskolin-treated HeLa cells transfected with the constructs W and M. Means±S.D. for three duplicate independent experiments are shown. ( B ) Effect of forskolin on the PiC mRNA level. RT–PCR was performed with specific primers for the cDNA of PiC or β-actin and with total RNA from HeLa cells treated or untreated with forskolin. ( C ) Effect of forskolin on the PiC protein content. The PiC protein of HeLa cells treated or untreated with forskolin was immunodecorated with an anti-N-terminal PiC antibody. The reactivity of the antibody to recombinant human PiC-B was also tested.

    Techniques Used: Activity Assay, Mutagenesis, Binding Assay, Transfection, Expressing, Construct, Reverse Transcription Polymerase Chain Reaction, Recombinant

    20) Product Images from "Identification and Characterization of ADNT1, a Novel Mitochondrial Adenine Nucleotide Transporter from Arabidopsis 1Identification and Characterization of ADNT1, a Novel Mitochondrial Adenine Nucleotide Transporter from Arabidopsis 1 [OA]"

    Article Title: Identification and Characterization of ADNT1, a Novel Mitochondrial Adenine Nucleotide Transporter from Arabidopsis 1Identification and Characterization of ADNT1, a Novel Mitochondrial Adenine Nucleotide Transporter from Arabidopsis 1 [OA]

    Journal: Plant Physiology

    doi: 10.1104/pp.108.130310

    Isolation and genetic characterization of an Arabidopsis mutant harboring a T-DNA insertion within the ADNT1 gene. A, Schematic representation of the ADNT1 gene. The mutant obtained by PCR screening of a T-DNA mutant collection (GABI-Kat) carries an insertion into the first intron of ADNT1. The scale of the T-DNA (approximately 4.5 kb) was not preserved for the sake of convenience. Boxes represent gene exons, and arrows on T-DNA, intron I, and intron IV denote primer positions for T2 and T3 population screening. B, PCR screening of the T2 population. M, DNA marker; T2, screened T2 population lines. Numbers at left indicate molecular masses in kilobases. C, Expression analysis of ADNT1. Northern blot of total RNA extracted from different tissues of the wild type (Col-0) and homozygous mutants (ADNT1 − /ADNT1 − ); le, leaf; st, stem; fl, flower. The bottom section shows rRNA as a loading control.
    Figure Legend Snippet: Isolation and genetic characterization of an Arabidopsis mutant harboring a T-DNA insertion within the ADNT1 gene. A, Schematic representation of the ADNT1 gene. The mutant obtained by PCR screening of a T-DNA mutant collection (GABI-Kat) carries an insertion into the first intron of ADNT1. The scale of the T-DNA (approximately 4.5 kb) was not preserved for the sake of convenience. Boxes represent gene exons, and arrows on T-DNA, intron I, and intron IV denote primer positions for T2 and T3 population screening. B, PCR screening of the T2 population. M, DNA marker; T2, screened T2 population lines. Numbers at left indicate molecular masses in kilobases. C, Expression analysis of ADNT1. Northern blot of total RNA extracted from different tissues of the wild type (Col-0) and homozygous mutants (ADNT1 − /ADNT1 − ); le, leaf; st, stem; fl, flower. The bottom section shows rRNA as a loading control.

    Techniques Used: Isolation, Mutagenesis, Polymerase Chain Reaction, Marker, Expressing, Northern Blot

    21) Product Images from "Vascular endothelial growth factor promoter‐based conditionally replicative adenoviruses effectively suppress growth of malignant pleural mesothelioma"

    Article Title: Vascular endothelial growth factor promoter‐based conditionally replicative adenoviruses effectively suppress growth of malignant pleural mesothelioma

    Journal: Cancer Science

    doi: 10.1111/cas.13112

    (a) Expression of VEGF m RNA in mesothelioma cell lines. A549, NCI ‐H157, and NCI ‐H358 are human lung cancer cell lines as positive control. MSTO ‐211H, NCI ‐H28, NCI ‐H226, NCI ‐H2052, and NCI ‐H2452 are human malignant mesothelioma cell lines. Each RT ‐ PCR product for VEGF 121 (408 bp) or VEGF 165 (541 bp) is shown in the upper panel, and that for GAPDH (574 bp) is shown in the lower panel. The intensity of the RT ‐ PCR band corresponding to VEGF 121 and VEGF 165 was quantitated using an image analyzer. (b) Luciferase activity in mesothelioma cell lines infected with Ad5 CMVL uc, Ad5 VEGFL uc, or Ad5/3 CMVL uc. 1 × 10 5 cells of each cell line were infected with each virus for 3 h at MOI 10. Cells were harvested and lysed in 100 μL of lysis buffer. 10 μL of each lysate was used for luciferase assay at 48 h after infection.
    Figure Legend Snippet: (a) Expression of VEGF m RNA in mesothelioma cell lines. A549, NCI ‐H157, and NCI ‐H358 are human lung cancer cell lines as positive control. MSTO ‐211H, NCI ‐H28, NCI ‐H226, NCI ‐H2052, and NCI ‐H2452 are human malignant mesothelioma cell lines. Each RT ‐ PCR product for VEGF 121 (408 bp) or VEGF 165 (541 bp) is shown in the upper panel, and that for GAPDH (574 bp) is shown in the lower panel. The intensity of the RT ‐ PCR band corresponding to VEGF 121 and VEGF 165 was quantitated using an image analyzer. (b) Luciferase activity in mesothelioma cell lines infected with Ad5 CMVL uc, Ad5 VEGFL uc, or Ad5/3 CMVL uc. 1 × 10 5 cells of each cell line were infected with each virus for 3 h at MOI 10. Cells were harvested and lysed in 100 μL of lysis buffer. 10 μL of each lysate was used for luciferase assay at 48 h after infection.

    Techniques Used: Expressing, Positive Control, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Infection, Lysis

    22) Product Images from "Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model"

    Article Title: Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9388

    Somatic chromosomal translocation between endogenous Ewsr1 and Fli1 loci in Model #12 Cre-TL-EF A . Targeting mouse embryonic stem cells to insert a lox-puromycin r -lox cassette between exons 8 and 9 of the Ewsr1 locus. Genomic DNAs from the embryonic stem cell clones were EcoRI/AgeI (left) or KpnI (right) digested and were analyzed for the 5’ and 3’ integrations using Southern blot. Green and purple horizontal bars represent the probes used in the Southern blots. B . Targeting mouse embryonic stem cells to insert a lox-hygromycin r -lox cassette between exons 5 and 6 of the Fli1 locus. Genomic DNAs from the embryonic stem cell clones were ApaI (left) or EcoRV/KpnI (right) digested and were analyzed for the 5’ and 3’ integrations using Southern blot. Green and purple bars represent the probes used in the Southern blots. C . Schematic illustration for adenoviral Cre infection of Ews lox/wt ; Fli1 lox/wt MEFs in vitro . Genomic PCR was used to detect the translocated and untranslocated Ews and Fli1 chromosomes. The locations of the primers used are presented in the schematic. D . qPCR for Ews-Fli1 on total RNA from adenoviral Cre-treated MEFs. Hprt was used as the control gene, and samples were normalized to uninfected MEFs.
    Figure Legend Snippet: Somatic chromosomal translocation between endogenous Ewsr1 and Fli1 loci in Model #12 Cre-TL-EF A . Targeting mouse embryonic stem cells to insert a lox-puromycin r -lox cassette between exons 8 and 9 of the Ewsr1 locus. Genomic DNAs from the embryonic stem cell clones were EcoRI/AgeI (left) or KpnI (right) digested and were analyzed for the 5’ and 3’ integrations using Southern blot. Green and purple horizontal bars represent the probes used in the Southern blots. B . Targeting mouse embryonic stem cells to insert a lox-hygromycin r -lox cassette between exons 5 and 6 of the Fli1 locus. Genomic DNAs from the embryonic stem cell clones were ApaI (left) or EcoRV/KpnI (right) digested and were analyzed for the 5’ and 3’ integrations using Southern blot. Green and purple bars represent the probes used in the Southern blots. C . Schematic illustration for adenoviral Cre infection of Ews lox/wt ; Fli1 lox/wt MEFs in vitro . Genomic PCR was used to detect the translocated and untranslocated Ews and Fli1 chromosomes. The locations of the primers used are presented in the schematic. D . qPCR for Ews-Fli1 on total RNA from adenoviral Cre-treated MEFs. Hprt was used as the control gene, and samples were normalized to uninfected MEFs.

    Techniques Used: Translocation Assay, Clone Assay, Southern Blot, Infection, In Vitro, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    23) Product Images from "Rapid and Sensitive Detection of Noroviruses by Using TaqMan-Based One-Step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples"

    Article Title: Rapid and Sensitive Detection of Noroviruses by Using TaqMan-Based One-Step Reverse Transcription-PCR Assays and Application to Naturally Contaminated Shellfish Samples

    Journal:

    doi: 10.1128/AEM.71.4.1870-1875.2005

    ), determined by gradient TaqMan RT-PCR with RNA from five different GII strains (lines 1, R4 [GII.1]; lines 2, c8 [GII.1]; lines 3, c9 [GII.2]; lines 4, B2
    Figure Legend Snippet: ), determined by gradient TaqMan RT-PCR with RNA from five different GII strains (lines 1, R4 [GII.1]; lines 2, c8 [GII.1]; lines 3, c9 [GII.2]; lines 4, B2

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    24) Product Images from "Postmenopausal Increase in KiSS-1, GPR54, and Luteinizing Hormone Releasing Hormone (LHRH-1) mRNA in the Basal Hypothalamus of Female Rhesus Monkeys"

    Article Title: Postmenopausal Increase in KiSS-1, GPR54, and Luteinizing Hormone Releasing Hormone (LHRH-1) mRNA in the Basal Hypothalamus of Female Rhesus Monkeys

    Journal:

    doi: 10.1016/j.peptides.2008.06.005

    A schematic illustration of tissue dissection from the monkey brain for quantitative RT-PCR. The mid sagittal section is shown. In this study, quantitative RT-PCR analysis was done on total RNA from POA (hatched) and MBH (dotted). Abbreviations: AA=anterior
    Figure Legend Snippet: A schematic illustration of tissue dissection from the monkey brain for quantitative RT-PCR. The mid sagittal section is shown. In this study, quantitative RT-PCR analysis was done on total RNA from POA (hatched) and MBH (dotted). Abbreviations: AA=anterior

    Techniques Used: Dissection, Quantitative RT-PCR

    25) Product Images from "TGF? signalling plays an important role in IL4-induced alternative activation of microglia"

    Article Title: TGF? signalling plays an important role in IL4-induced alternative activation of microglia

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-9-210

    Arg1 and Ym1 expression induced by IL4 was blocked in the presence of a TGFβ receptor type I inhibitor. Primary microglia were treated with IL4 (10 ng/ml) combined either with or without TGFβ receptor type I kinase inhibitor IV (TβKI, 2 μM) for 24 hours. RNA and proteins were isolated for quantitative RT-PCR and western blotting, respectively. Quantitative RT-PCR shows that IL4 treatment significantly increased Arg1 ( A ) and Ym1 ( B ) mRNA levels ( P
    Figure Legend Snippet: Arg1 and Ym1 expression induced by IL4 was blocked in the presence of a TGFβ receptor type I inhibitor. Primary microglia were treated with IL4 (10 ng/ml) combined either with or without TGFβ receptor type I kinase inhibitor IV (TβKI, 2 μM) for 24 hours. RNA and proteins were isolated for quantitative RT-PCR and western blotting, respectively. Quantitative RT-PCR shows that IL4 treatment significantly increased Arg1 ( A ) and Ym1 ( B ) mRNA levels ( P

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Western Blot

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    SYBR Green Assay:

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    Article Snippet: .. The amount of 1 μg of RNA was reverse transcribed to cDNA using random hexamer primers from GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA) in a 20 μL reaction volume at 42o C for 30 min. Quantification of PCR products was performed in 10 μL of Lightcycler 480 SYBR Green I Master mix (Roche) using a real-time PCR Lightcycler (Roche). .. DNA was denatured at 95o C for 2 min; then followed 45 cycles of denaturation at 95o C for 25 s, annealing at 60o C for 45 s, elongation at 72o C for 1 min and incubation at 80o C for 5 s. cDNAs were amplified with specific primers for β-actin, TAP-1, LMP-2, TAP-2, and LMP-7 .

    Negative Control:

    Article Title: Regulatory Role of the MisR/S Two-Component System in Hemoglobin Utilization in Neisseria meningitidis ▿ ▿ †
    Article Snippet: .. PCR amplification of the RNA samples using primers hmbR-p-f and hmbR-p-r confirmed that there was no genomic DNA contamination. cDNA was obtained by reverse transcription (RT) of total RNA (1 μg) using a GeneAmp RNA PCR core kit (Applied Biosystems), and reactions without the reverse transcriptase were used as a negative control. .. The transcription of genes of interest was measured by real-time quantitative RT (qRT)-PCR using the SYBR green detection method (SYBR green Supermix; Bio-Rad); the reaction conditions used have been described previously ( ).

    Amplification:

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    Article Title: Regulatory Role of the MisR/S Two-Component System in Hemoglobin Utilization in Neisseria meningitidis ▿ ▿ †
    Article Snippet: .. PCR amplification of the RNA samples using primers hmbR-p-f and hmbR-p-r confirmed that there was no genomic DNA contamination. cDNA was obtained by reverse transcription (RT) of total RNA (1 μg) using a GeneAmp RNA PCR core kit (Applied Biosystems), and reactions without the reverse transcriptase were used as a negative control. .. The transcription of genes of interest was measured by real-time quantitative RT (qRT)-PCR using the SYBR green detection method (SYBR green Supermix; Bio-Rad); the reaction conditions used have been described previously ( ).

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    Synthesized:

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    Article Snippet: .. Real-time quantitative PCR cDNA was synthesized from 5 μg of total RNA from each sample using the GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA, USA). .. The cDNA were amplified with KAPA SYBR FAST Universal qPCR kit (Kapa Biosystems, Wilmington, MA, USA) using the following primers (Additional file : Table S1).

    Real-time Polymerase Chain Reaction:

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    Article Title: Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling
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    Polymerase Chain Reaction:

    Article Title: Treatment of endometriosis with a VEGF targeted conditionally replicative adenovirus
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    Random Hexamer Labeling:

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    Reverse Transcription Polymerase Chain Reaction:

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