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MACHEREY NAGEL real time pcr total rna extraction
Real Time Pcr Total Rna Extraction, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Real-time Polymerase Chain Reaction:

Article Title: Effect of Supplementing Hydrolysable Tannins to a Grower–Finisher Diet Containing Divergent PUFA Levels on Growth Performance, Boar Taint Levels in Back Fat and Intestinal Microbiota of Entire Males
Article Snippet: .. RNA Isolation, Primer Design, and Quantitative Real-Time PCR Total RNA extraction from the liver and colon mucosa was performed using Nucleospin® RNA XS kit (740902, Macherey–Nagel, Oensinger, Switzerland) as previously described [ ]. .. Concentrations were determined using a Nanodrop kit (Witec, Luzern, Switzerland) and quality assessed by capillary electrophoresis using a Fragment Analyzer (Advanced Instruments, Norwood, United States).

Article Title: Apigenin enhances skeletal muscle hypertrophy and myoblast differentiation by regulating Prmt7
Article Snippet: .. RNA extraction and real-time PCR Total RNA extraction was performed using NucleoSpin RNAII(Macherey-Nagel, Düren, Germany), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). .. Quantitative real-time PCR was performed with the SYBR Green Master Mix (Toyobo, Osaka, Japan).

Article Title: Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution
Article Snippet: .. 2.7 RNA extraction and Real-time PCR Total RNA extraction from the renal cortex and medulla was performed using NucleoSpin RNA II RNA extraction kit (Macherey-Nagel). .. RNA was reversed transcribed using qScript cDNA Super Mix (Quanta Biosciences).10 ng of cDNA was used for the PCR reaction with SYBR Green Master Mix (Applied Biosystems) and the Real-time PCR reaction was performed in duplicates using the StepOne Plus Real-Time PCR System (AB Applied Biosystems) [ , ].

Isolation:

Article Title: Effect of Supplementing Hydrolysable Tannins to a Grower–Finisher Diet Containing Divergent PUFA Levels on Growth Performance, Boar Taint Levels in Back Fat and Intestinal Microbiota of Entire Males
Article Snippet: .. RNA Isolation, Primer Design, and Quantitative Real-Time PCR Total RNA extraction from the liver and colon mucosa was performed using Nucleospin® RNA XS kit (740902, Macherey–Nagel, Oensinger, Switzerland) as previously described [ ]. .. Concentrations were determined using a Nanodrop kit (Witec, Luzern, Switzerland) and quality assessed by capillary electrophoresis using a Fragment Analyzer (Advanced Instruments, Norwood, United States).

RNA Extraction:

Article Title: Effect of Supplementing Hydrolysable Tannins to a Grower–Finisher Diet Containing Divergent PUFA Levels on Growth Performance, Boar Taint Levels in Back Fat and Intestinal Microbiota of Entire Males
Article Snippet: .. RNA Isolation, Primer Design, and Quantitative Real-Time PCR Total RNA extraction from the liver and colon mucosa was performed using Nucleospin® RNA XS kit (740902, Macherey–Nagel, Oensinger, Switzerland) as previously described [ ]. .. Concentrations were determined using a Nanodrop kit (Witec, Luzern, Switzerland) and quality assessed by capillary electrophoresis using a Fragment Analyzer (Advanced Instruments, Norwood, United States).

Article Title: Apigenin enhances skeletal muscle hypertrophy and myoblast differentiation by regulating Prmt7
Article Snippet: .. RNA extraction and real-time PCR Total RNA extraction was performed using NucleoSpin RNAII(Macherey-Nagel, Düren, Germany), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). .. Quantitative real-time PCR was performed with the SYBR Green Master Mix (Toyobo, Osaka, Japan).

Article Title: Tubular NOX4 expression decreases in chronic kidney disease but does not modify fibrosis evolution
Article Snippet: .. 2.7 RNA extraction and Real-time PCR Total RNA extraction from the renal cortex and medulla was performed using NucleoSpin RNA II RNA extraction kit (Macherey-Nagel). .. RNA was reversed transcribed using qScript cDNA Super Mix (Quanta Biosciences).10 ng of cDNA was used for the PCR reaction with SYBR Green Master Mix (Applied Biosystems) and the Real-time PCR reaction was performed in duplicates using the StepOne Plus Real-Time PCR System (AB Applied Biosystems) [ , ].

Synthesized:

Article Title: Apigenin enhances skeletal muscle hypertrophy and myoblast differentiation by regulating Prmt7
Article Snippet: .. RNA extraction and real-time PCR Total RNA extraction was performed using NucleoSpin RNAII(Macherey-Nagel, Düren, Germany), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). .. Quantitative real-time PCR was performed with the SYBR Green Master Mix (Toyobo, Osaka, Japan).

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    MACHEREY NAGEL total rnas
    Overexpression of SAMHD1 reduces extracellular and intracellular levels of HBV DNA in HepG2.2.15 cells without affecting viral <t>RNA.</t> HepG2.2.15 cells were transfected with ( a ) increasing amounts or ( b – d ) 1 μg of a SAMHD1-coding plasmid. The total amount of plasmid DNA was adjusted to 1 μg by addition of empty vector. Twenty-four hours after transfection, the cells were washed with phosphate-buffered saline (PBS) and further cultured in serum-free medium without supplements. Forty-eight hours later, HBV DNA from the supernatant ( a , d ) and intracellular HBV DNA from cytoplasmic fractions ( c ) were quantified by qPCR. ( b ) The levels of intracellular HBV <t>RNAs</t> were determined by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( a ) The mean ± SEM of the technical triplicates is shown. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p
    Total Rnas, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 93/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total rnas/product/MACHEREY NAGEL
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    MACHEREY NAGEL real time rt pcr total rna
    Stable expression of HS3ST3B in MDA-MB-231 cells. Cells were transfected with the expression vector encoding HS3ST3B and then cultured in complete DMEM medium in the presence of 400 µg/mL G418. After 14 days of culture, individual colonies were isolated by limit dilution and amplified in medium supplemented with G418. In parallel, cells were transfected with the empty vector to obtain the control parental cells (pEmpty). ( A ) Following <t>RNA</t> extraction, the mRNA level of HS3ST3B was quantified by real-time <t>RT-PCR</t> in each clone. Relative abundance of the transcripts was normalized to endogenous HPRT mRNA. Data are means ± SD of triplicates. ( B ) HS3ST3B expression in the clones C and D was analyzed by confocal microscopy. To this end, cells were seeded on glass coverslips, permeabilized and then incubated in the presence of anti-HS3ST3B antibodies. After wash, they were immunostained with secondary antibodies conjugated to Alexa-568, in order to highlight the enzyme in red fluorescence. For the detection of 3- O -sulfated motifs, recombinant HSV-1 gD (10 μg/mL) was incubated with primary anti-gD antibody for 30 min at 4 °C, and the immune complex was added to cells for an additional 30 min-incubation. After washing, cells were fixed and incubated for 1 h with Alexa 488-conjugated secondary antibody (green fluorescence). In all the microscopy experiments, nuclei were stained in blue with DAPI, in order to visualize cell nuclei ( N = 3 separate experiments; n = 30 cells). Scale bar = 10 µm.
    Real Time Rt Pcr Total Rna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL reverse transcription quantitative polymerase chain reaction total rna
    The porcine reproductive and respiratory syndrome virus (PRRSV) <t>Lena</t> strain induces IFN-α in infected animals but cannot directly activate pDC in vitro . (A) Serum IFN-α responses of pigs infected oronasally with PRRSV Lena (5 × 10 5 TCID 50 per animal), as determined by ELISA. (B) Viral <t>RNA</t> loads in the serum. The data was obtained using RT-qPCR for PRRSV ORF7 and is represented as the total number of cycles minus the quantification cycle (Cq)-value. (C) Comparative analysis of IFN-α production by enriched pDC in response to PRRSV-1 (LVP-23 and Lena) and PRRSV-2 strains (VR-2332 and RVB-581) after stimulation with a MOI of 0.1 TCID 50 /cell. CpG D32 was included as control. The supernatants were harvested after 20 h of incubation and IFN-α was quantified by ELISA. The data represent the mean IFN-α (U/ml) of triplicate cultures, with error bars representing the standard deviation. These results are representative of at least three independent experiments.
    Reverse Transcription Quantitative Polymerase Chain Reaction Total Rna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL real time qrt pcr total rna
    Inhibitory effect of MUPCDH on cell proliferation. ( a ) Knockdown of the MUPCDH gene increased cell survival in human renal cortical epithelial (HRCE) cells. ( b ) Overexpression of the MUPCDH gene decreased cell growth of WT9-7 cells. ( c ) 5-bromo-2′-deoxyuridine (BrdU) incorporation assays were carried out using HRCE cells that had been transfected with MUPCDH short interfering <t>RNA</t> (siRNA) or control siRNA. ( d ) Proliferating cell nuclear antigen (PCNA) staining carried out using HEK293T cells that overexpressed the MUPCDH gene. The level of PCNA expression was also measured by real-time quantitative reverse transcription polymerase chain reaction <t>(qRT-PCR).</t> β-Actin was used as an internal control in real-time qRT-PCR assays. ( e ) Cell proliferation rate was measured using a cell proliferation assay kit (WST-1) following treatment of WT9-7 cells with 5-aza-2′-deoxycytidine (5 μM). The experiment was performed in triplicate. * P
    Real Time Qrt Pcr Total Rna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overexpression of SAMHD1 reduces extracellular and intracellular levels of HBV DNA in HepG2.2.15 cells without affecting viral RNA. HepG2.2.15 cells were transfected with ( a ) increasing amounts or ( b – d ) 1 μg of a SAMHD1-coding plasmid. The total amount of plasmid DNA was adjusted to 1 μg by addition of empty vector. Twenty-four hours after transfection, the cells were washed with phosphate-buffered saline (PBS) and further cultured in serum-free medium without supplements. Forty-eight hours later, HBV DNA from the supernatant ( a , d ) and intracellular HBV DNA from cytoplasmic fractions ( c ) were quantified by qPCR. ( b ) The levels of intracellular HBV RNAs were determined by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( a ) The mean ± SEM of the technical triplicates is shown. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Journal: Scientific Reports

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

    doi: 10.1038/srep26616

    Figure Lengend Snippet: Overexpression of SAMHD1 reduces extracellular and intracellular levels of HBV DNA in HepG2.2.15 cells without affecting viral RNA. HepG2.2.15 cells were transfected with ( a ) increasing amounts or ( b – d ) 1 μg of a SAMHD1-coding plasmid. The total amount of plasmid DNA was adjusted to 1 μg by addition of empty vector. Twenty-four hours after transfection, the cells were washed with phosphate-buffered saline (PBS) and further cultured in serum-free medium without supplements. Forty-eight hours later, HBV DNA from the supernatant ( a , d ) and intracellular HBV DNA from cytoplasmic fractions ( c ) were quantified by qPCR. ( b ) The levels of intracellular HBV RNAs were determined by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( a ) The mean ± SEM of the technical triplicates is shown. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Article Snippet: RNA extraction and reverse transcription quantitative PCR analysis For RT-qPCR analysis, total RNAs were extracted from cell pellets using NucleoSpin® RNA Plus Kit (Macherey Nagel).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    dATP levels are affected by HBV infection. SAMHD1 is de-phosphorylated at T592 in primary human hepatocytes, and is not affected by HBV infection. ( a ) HepG2-NTCP- control-shRNA cells were infected or not with HBV in presence of 2.5%DMSO and dATP content was measured 4 days post infection. The mean ± SEM of technical duplicates is represented. ( b , c ) Primary human hepatocytes (PHH) from one donnor were infected or not with HBV for 8 or 24 hours. ( b ) SAMHD1 phosphorylation on T592, total SAMHD1, cyclin B1 and GAPDH were detected by Western blotting. Cycling or serum starved HepG2.2.15-control-shRNA cells were used respectively as positive and negative controls for pT592-SAMHD1 and cyclin B1. A representative experiment out of two technical replicates is represented. ( c ) Levels of intracellular HBV RNAs from infected PHH used in ( b ) were determined by RT-qPCR using HBV primer set 3 (see Fig. 2b and Table 1 ; amplified HBV RNA species: 3.5 kb, 2.4 kb and 2.1 kb RNAs). B.d.: below detection. The mean ± SEM of technical triplicates is represented.

    Journal: Scientific Reports

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

    doi: 10.1038/srep26616

    Figure Lengend Snippet: dATP levels are affected by HBV infection. SAMHD1 is de-phosphorylated at T592 in primary human hepatocytes, and is not affected by HBV infection. ( a ) HepG2-NTCP- control-shRNA cells were infected or not with HBV in presence of 2.5%DMSO and dATP content was measured 4 days post infection. The mean ± SEM of technical duplicates is represented. ( b , c ) Primary human hepatocytes (PHH) from one donnor were infected or not with HBV for 8 or 24 hours. ( b ) SAMHD1 phosphorylation on T592, total SAMHD1, cyclin B1 and GAPDH were detected by Western blotting. Cycling or serum starved HepG2.2.15-control-shRNA cells were used respectively as positive and negative controls for pT592-SAMHD1 and cyclin B1. A representative experiment out of two technical replicates is represented. ( c ) Levels of intracellular HBV RNAs from infected PHH used in ( b ) were determined by RT-qPCR using HBV primer set 3 (see Fig. 2b and Table 1 ; amplified HBV RNA species: 3.5 kb, 2.4 kb and 2.1 kb RNAs). B.d.: below detection. The mean ± SEM of technical triplicates is represented.

    Article Snippet: RNA extraction and reverse transcription quantitative PCR analysis For RT-qPCR analysis, total RNAs were extracted from cell pellets using NucleoSpin® RNA Plus Kit (Macherey Nagel).

    Techniques: Infection, shRNA, Western Blot, Quantitative RT-PCR, Amplification

    SAMHD1 restricts HBV replication in infected HepG2-NTCP cells. Detection of ( a ) SAMHD1 or ( b ) NTCP in SAMHD1 knockdown or control HepG2-NTCP cells by Western blotting. Actin or GAPDH were used as loading controls. ( c ) Equal cell growth and viability was assessed was assessed using ATPLite. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( d ) Control shRNA cells were cultured for 10 days in complete medium supplemented (+) or not (−) with 2.5% DMSO. When indicated cells were infected with HBV 48 hours after start of DMSO treatment. Phosphorylation at residue T592 in SAMHD1, as well as expression of total SAMHD1, cyclin B1 and actin, were detected by Western blotting. ( e ) Cells were cultured for 10 days in 2.5% DMSO-containing medium and infected with a single-round luciferase HIV-1 virus. Luciferase activity was detected 24 hours post infection. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( f-i ) Cells were infected with HBV inoculum, and where indicated, the entry inhibitor MyrcludexB (MyrB, 200 nM), or the reverse transcription inhibitor lamivudine (Lami, 0,5 μM) were added. Cells were cultured in medium containing 2.5% DMSO. Ten days post infection, HBV DNA from the supernatant ( f ), intracellular total HBV DNA ( g ) and cccDNA ( h ) were quantified by qPCR. ( i ) The intracellular HBV RNAs levels were determined 10 days post infection by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( f ) For each individual experiment, fold-changes to the untreated control shRNA were calculated based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is depicted. ( g – i ) For each independent experiment, fold-changes to untreated control shRNA were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of at least 3 independent experiments is represented. ( f – i ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Journal: Scientific Reports

    Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle

    doi: 10.1038/srep26616

    Figure Lengend Snippet: SAMHD1 restricts HBV replication in infected HepG2-NTCP cells. Detection of ( a ) SAMHD1 or ( b ) NTCP in SAMHD1 knockdown or control HepG2-NTCP cells by Western blotting. Actin or GAPDH were used as loading controls. ( c ) Equal cell growth and viability was assessed was assessed using ATPLite. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( d ) Control shRNA cells were cultured for 10 days in complete medium supplemented (+) or not (−) with 2.5% DMSO. When indicated cells were infected with HBV 48 hours after start of DMSO treatment. Phosphorylation at residue T592 in SAMHD1, as well as expression of total SAMHD1, cyclin B1 and actin, were detected by Western blotting. ( e ) Cells were cultured for 10 days in 2.5% DMSO-containing medium and infected with a single-round luciferase HIV-1 virus. Luciferase activity was detected 24 hours post infection. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( f-i ) Cells were infected with HBV inoculum, and where indicated, the entry inhibitor MyrcludexB (MyrB, 200 nM), or the reverse transcription inhibitor lamivudine (Lami, 0,5 μM) were added. Cells were cultured in medium containing 2.5% DMSO. Ten days post infection, HBV DNA from the supernatant ( f ), intracellular total HBV DNA ( g ) and cccDNA ( h ) were quantified by qPCR. ( i ) The intracellular HBV RNAs levels were determined 10 days post infection by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( f ) For each individual experiment, fold-changes to the untreated control shRNA were calculated based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is depicted. ( g – i ) For each independent experiment, fold-changes to untreated control shRNA were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of at least 3 independent experiments is represented. ( f – i ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p

    Article Snippet: RNA extraction and reverse transcription quantitative PCR analysis For RT-qPCR analysis, total RNAs were extracted from cell pellets using NucleoSpin® RNA Plus Kit (Macherey Nagel).

    Techniques: Infection, Western Blot, shRNA, Cell Culture, Expressing, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Stable expression of HS3ST3B in MDA-MB-231 cells. Cells were transfected with the expression vector encoding HS3ST3B and then cultured in complete DMEM medium in the presence of 400 µg/mL G418. After 14 days of culture, individual colonies were isolated by limit dilution and amplified in medium supplemented with G418. In parallel, cells were transfected with the empty vector to obtain the control parental cells (pEmpty). ( A ) Following RNA extraction, the mRNA level of HS3ST3B was quantified by real-time RT-PCR in each clone. Relative abundance of the transcripts was normalized to endogenous HPRT mRNA. Data are means ± SD of triplicates. ( B ) HS3ST3B expression in the clones C and D was analyzed by confocal microscopy. To this end, cells were seeded on glass coverslips, permeabilized and then incubated in the presence of anti-HS3ST3B antibodies. After wash, they were immunostained with secondary antibodies conjugated to Alexa-568, in order to highlight the enzyme in red fluorescence. For the detection of 3- O -sulfated motifs, recombinant HSV-1 gD (10 μg/mL) was incubated with primary anti-gD antibody for 30 min at 4 °C, and the immune complex was added to cells for an additional 30 min-incubation. After washing, cells were fixed and incubated for 1 h with Alexa 488-conjugated secondary antibody (green fluorescence). In all the microscopy experiments, nuclei were stained in blue with DAPI, in order to visualize cell nuclei ( N = 3 separate experiments; n = 30 cells). Scale bar = 10 µm.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: The Pro-Tumoral Activity of Heparan Sulfate 3-O-Sulfotransferase 3B (HS3ST3B) in Breast Cancer MDA-MB-231 Cells Is Dependent on the Expression of Neuropilin-1

    doi: 10.3390/molecules23102718

    Figure Lengend Snippet: Stable expression of HS3ST3B in MDA-MB-231 cells. Cells were transfected with the expression vector encoding HS3ST3B and then cultured in complete DMEM medium in the presence of 400 µg/mL G418. After 14 days of culture, individual colonies were isolated by limit dilution and amplified in medium supplemented with G418. In parallel, cells were transfected with the empty vector to obtain the control parental cells (pEmpty). ( A ) Following RNA extraction, the mRNA level of HS3ST3B was quantified by real-time RT-PCR in each clone. Relative abundance of the transcripts was normalized to endogenous HPRT mRNA. Data are means ± SD of triplicates. ( B ) HS3ST3B expression in the clones C and D was analyzed by confocal microscopy. To this end, cells were seeded on glass coverslips, permeabilized and then incubated in the presence of anti-HS3ST3B antibodies. After wash, they were immunostained with secondary antibodies conjugated to Alexa-568, in order to highlight the enzyme in red fluorescence. For the detection of 3- O -sulfated motifs, recombinant HSV-1 gD (10 μg/mL) was incubated with primary anti-gD antibody for 30 min at 4 °C, and the immune complex was added to cells for an additional 30 min-incubation. After washing, cells were fixed and incubated for 1 h with Alexa 488-conjugated secondary antibody (green fluorescence). In all the microscopy experiments, nuclei were stained in blue with DAPI, in order to visualize cell nuclei ( N = 3 separate experiments; n = 30 cells). Scale bar = 10 µm.

    Article Snippet: RNA Isolation and Real-Time RT-PCR Total RNA was isolated from 2 × 105 cells using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Cell Culture, Isolation, Amplification, RNA Extraction, Quantitative RT-PCR, Clone Assay, Confocal Microscopy, Incubation, Fluorescence, Recombinant, Microscopy, Staining

    Knockdown of the expression of Nrp1 in MDA-MB-231 cells by RNA interference. Cells were transfected with the negative control siRNA (siCtrl) or specific siRNA targeting Nrp1 (siNrp1). ( A ) After 24 h of treatment, the levels of mRNA encoding Nrp1 were determined by real-time RT-PCR. Relative abundance of the transcripts was normalized to endogenous HPRT mRNA. Data are means ± SD of triplicates. ( B ) The efficacy of specific siRNA to knockdown the expression of Nrp1 was verified by Western blot 48 h post-transfection. Parallel immunoblotting with anti-GAPDH confirmed equal loading of the samples. Data are representative of three independent experiments.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: The Pro-Tumoral Activity of Heparan Sulfate 3-O-Sulfotransferase 3B (HS3ST3B) in Breast Cancer MDA-MB-231 Cells Is Dependent on the Expression of Neuropilin-1

    doi: 10.3390/molecules23102718

    Figure Lengend Snippet: Knockdown of the expression of Nrp1 in MDA-MB-231 cells by RNA interference. Cells were transfected with the negative control siRNA (siCtrl) or specific siRNA targeting Nrp1 (siNrp1). ( A ) After 24 h of treatment, the levels of mRNA encoding Nrp1 were determined by real-time RT-PCR. Relative abundance of the transcripts was normalized to endogenous HPRT mRNA. Data are means ± SD of triplicates. ( B ) The efficacy of specific siRNA to knockdown the expression of Nrp1 was verified by Western blot 48 h post-transfection. Parallel immunoblotting with anti-GAPDH confirmed equal loading of the samples. Data are representative of three independent experiments.

    Article Snippet: RNA Isolation and Real-Time RT-PCR Total RNA was isolated from 2 × 105 cells using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Negative Control, Quantitative RT-PCR, Western Blot

    The porcine reproductive and respiratory syndrome virus (PRRSV) Lena strain induces IFN-α in infected animals but cannot directly activate pDC in vitro . (A) Serum IFN-α responses of pigs infected oronasally with PRRSV Lena (5 × 10 5 TCID 50 per animal), as determined by ELISA. (B) Viral RNA loads in the serum. The data was obtained using RT-qPCR for PRRSV ORF7 and is represented as the total number of cycles minus the quantification cycle (Cq)-value. (C) Comparative analysis of IFN-α production by enriched pDC in response to PRRSV-1 (LVP-23 and Lena) and PRRSV-2 strains (VR-2332 and RVB-581) after stimulation with a MOI of 0.1 TCID 50 /cell. CpG D32 was included as control. The supernatants were harvested after 20 h of incubation and IFN-α was quantified by ELISA. The data represent the mean IFN-α (U/ml) of triplicate cultures, with error bars representing the standard deviation. These results are representative of at least three independent experiments.

    Journal: Frontiers in Microbiology

    Article Title: Sensing of Porcine Reproductive and Respiratory Syndrome Virus-Infected Macrophages by Plasmacytoid Dendritic Cells

    doi: 10.3389/fmicb.2016.00771

    Figure Lengend Snippet: The porcine reproductive and respiratory syndrome virus (PRRSV) Lena strain induces IFN-α in infected animals but cannot directly activate pDC in vitro . (A) Serum IFN-α responses of pigs infected oronasally with PRRSV Lena (5 × 10 5 TCID 50 per animal), as determined by ELISA. (B) Viral RNA loads in the serum. The data was obtained using RT-qPCR for PRRSV ORF7 and is represented as the total number of cycles minus the quantification cycle (Cq)-value. (C) Comparative analysis of IFN-α production by enriched pDC in response to PRRSV-1 (LVP-23 and Lena) and PRRSV-2 strains (VR-2332 and RVB-581) after stimulation with a MOI of 0.1 TCID 50 /cell. CpG D32 was included as control. The supernatants were harvested after 20 h of incubation and IFN-α was quantified by ELISA. The data represent the mean IFN-α (U/ml) of triplicate cultures, with error bars representing the standard deviation. These results are representative of at least three independent experiments.

    Article Snippet: RNA Extraction and Reverse Transcription Quantitative Polymerase Chain Reaction Total RNA from serum of Lena-infected pigs and PRRSV-infected MØ supernatant was extracted using the Nucleospin RNA II kit (Macherey-Nagel AG, Oensingen, Switzerland) following the manufacturer’s instructions, including DNase treatment and DNase inactivation steps.

    Techniques: Infection, In Vitro, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Incubation, Standard Deviation

    Inhibitory effect of MUPCDH on cell proliferation. ( a ) Knockdown of the MUPCDH gene increased cell survival in human renal cortical epithelial (HRCE) cells. ( b ) Overexpression of the MUPCDH gene decreased cell growth of WT9-7 cells. ( c ) 5-bromo-2′-deoxyuridine (BrdU) incorporation assays were carried out using HRCE cells that had been transfected with MUPCDH short interfering RNA (siRNA) or control siRNA. ( d ) Proliferating cell nuclear antigen (PCNA) staining carried out using HEK293T cells that overexpressed the MUPCDH gene. The level of PCNA expression was also measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). β-Actin was used as an internal control in real-time qRT-PCR assays. ( e ) Cell proliferation rate was measured using a cell proliferation assay kit (WST-1) following treatment of WT9-7 cells with 5-aza-2′-deoxycytidine (5 μM). The experiment was performed in triplicate. * P

    Journal: Scientific Reports

    Article Title: Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD

    doi: 10.1038/srep15238

    Figure Lengend Snippet: Inhibitory effect of MUPCDH on cell proliferation. ( a ) Knockdown of the MUPCDH gene increased cell survival in human renal cortical epithelial (HRCE) cells. ( b ) Overexpression of the MUPCDH gene decreased cell growth of WT9-7 cells. ( c ) 5-bromo-2′-deoxyuridine (BrdU) incorporation assays were carried out using HRCE cells that had been transfected with MUPCDH short interfering RNA (siRNA) or control siRNA. ( d ) Proliferating cell nuclear antigen (PCNA) staining carried out using HEK293T cells that overexpressed the MUPCDH gene. The level of PCNA expression was also measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). β-Actin was used as an internal control in real-time qRT-PCR assays. ( e ) Cell proliferation rate was measured using a cell proliferation assay kit (WST-1) following treatment of WT9-7 cells with 5-aza-2′-deoxycytidine (5 μM). The experiment was performed in triplicate. * P

    Article Snippet: Total RNA extraction and real-time qRT-PCR Total RNA was extracted using the NucleoSpin® TriPrep Extract kit (MACHEREY-NAGEL), according to the manufacturer’s protocol.

    Techniques: Over Expression, BrdU Incorporation Assay, Transfection, Small Interfering RNA, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Proliferation Assay

    Downregulation of MUPCDH in ADPKD. ( a,b ) Gene expression levels were confirmed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses, in non-ADPKD and ADPKD renal cortical tissue, and in ( c,d ) human renal cortical epithelial and renal cystic epithelial (WT9-7) cells. The band density in western blot analysis was measured using the MultiGauge software. The height of each bar represents the mean, and the error bars indicate ± SD. Aquaporin 1 (AQP1), which is a proximal tubule marker, and β-Actin were used as internal controls in real-time qRT-PCR and western blot analyses. Each experiment was performed in triplicate. * P

    Journal: Scientific Reports

    Article Title: Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD

    doi: 10.1038/srep15238

    Figure Lengend Snippet: Downregulation of MUPCDH in ADPKD. ( a,b ) Gene expression levels were confirmed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses, in non-ADPKD and ADPKD renal cortical tissue, and in ( c,d ) human renal cortical epithelial and renal cystic epithelial (WT9-7) cells. The band density in western blot analysis was measured using the MultiGauge software. The height of each bar represents the mean, and the error bars indicate ± SD. Aquaporin 1 (AQP1), which is a proximal tubule marker, and β-Actin were used as internal controls in real-time qRT-PCR and western blot analyses. Each experiment was performed in triplicate. * P

    Article Snippet: Total RNA extraction and real-time qRT-PCR Total RNA was extracted using the NucleoSpin® TriPrep Extract kit (MACHEREY-NAGEL), according to the manufacturer’s protocol.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Software, Marker

    The regulation of MUPCDH transcription via transcription factor AP-2α. ( a ) As assayed by chromatin immunoprecipitation, together with quantitative reverse transcription polymerase chain reaction (qRT-PCR), AP-2α was highly enriched in the MUPCDH promoter region of human renal cortical epithelial cells. RNA polymerase II was used as a positive control. ( b , c ) Knockdown of AP-2α decreased the mRNA and protein expression levels of MUPCDH , as confirmed by real-time qRT-PCR and western blot analyses, respectively. The band density was measured using the MultiGauge software. ( d ) The luciferase activity was measured following knockdown of AP-2α in HEK293T cells. The ratio of Renilla luciferase to Firefly luciferase was calculated for each experiment, and the values from triplicate experiments were averaged. The mean value for each test construct was normalized to the activity of the empty vector. Bars represent the mean of the normalized values with error bars indicating the range. Each experiment was performed in triplicate. * P

    Journal: Scientific Reports

    Article Title: Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD

    doi: 10.1038/srep15238

    Figure Lengend Snippet: The regulation of MUPCDH transcription via transcription factor AP-2α. ( a ) As assayed by chromatin immunoprecipitation, together with quantitative reverse transcription polymerase chain reaction (qRT-PCR), AP-2α was highly enriched in the MUPCDH promoter region of human renal cortical epithelial cells. RNA polymerase II was used as a positive control. ( b , c ) Knockdown of AP-2α decreased the mRNA and protein expression levels of MUPCDH , as confirmed by real-time qRT-PCR and western blot analyses, respectively. The band density was measured using the MultiGauge software. ( d ) The luciferase activity was measured following knockdown of AP-2α in HEK293T cells. The ratio of Renilla luciferase to Firefly luciferase was calculated for each experiment, and the values from triplicate experiments were averaged. The mean value for each test construct was normalized to the activity of the empty vector. Bars represent the mean of the normalized values with error bars indicating the range. Each experiment was performed in triplicate. * P

    Article Snippet: Total RNA extraction and real-time qRT-PCR Total RNA was extracted using the NucleoSpin® TriPrep Extract kit (MACHEREY-NAGEL), according to the manufacturer’s protocol.

    Techniques: Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Positive Control, Expressing, Western Blot, Software, Luciferase, Activity Assay, Construct, Plasmid Preparation