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  • 94
    Thermo Fisher arraycontrol rna spikes
    Arraycontrol Rna Spikes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna targets
    Serum shock is sufficient to synchronize circadian gene expression in C2C12 myotubes. Stable clones of C2C12 cells (CE-4.7 MyoD ) were differentiated for 5 days prior to serum shock. <t>RNA</t> was isolated at the indicated time post-serum shock and used to determine the expression level of Bmal1 and MyoD by <t>RT-PCR.</t> The target transcript levels were normalized to Rpl26 transcript levels and then normalized to the highest point across all time points. The data for the graphs represent the mean values ± SEM for three independent clones for the reporter gene. The expression of endogenous Bmal1 ( A ), endogenous MyoD ( B ) all displayed a circadian oscillation as indicated by significant differences in peak and trough expression. For each respective gene, one-way ANOVA followed by Tukey post-hoc test identified significant difference ( P
    Rna Targets, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 18s rna
    Serum shock is sufficient to synchronize circadian gene expression in C2C12 myotubes. Stable clones of C2C12 cells (CE-4.7 MyoD ) were differentiated for 5 days prior to serum shock. <t>RNA</t> was isolated at the indicated time post-serum shock and used to determine the expression level of Bmal1 and MyoD by <t>RT-PCR.</t> The target transcript levels were normalized to Rpl26 transcript levels and then normalized to the highest point across all time points. The data for the graphs represent the mean values ± SEM for three independent clones for the reporter gene. The expression of endogenous Bmal1 ( A ), endogenous MyoD ( B ) all displayed a circadian oscillation as indicated by significant differences in peak and trough expression. For each respective gene, one-way ANOVA followed by Tukey post-hoc test identified significant difference ( P
    18s Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 993 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nucleolar rna rnu48
    Serum shock is sufficient to synchronize circadian gene expression in C2C12 myotubes. Stable clones of C2C12 cells (CE-4.7 MyoD ) were differentiated for 5 days prior to serum shock. <t>RNA</t> was isolated at the indicated time post-serum shock and used to determine the expression level of Bmal1 and MyoD by <t>RT-PCR.</t> The target transcript levels were normalized to Rpl26 transcript levels and then normalized to the highest point across all time points. The data for the graphs represent the mean values ± SEM for three independent clones for the reporter gene. The expression of endogenous Bmal1 ( A ), endogenous MyoD ( B ) all displayed a circadian oscillation as indicated by significant differences in peak and trough expression. For each respective gene, one-way ANOVA followed by Tukey post-hoc test identified significant difference ( P
    Nucleolar Rna Rnu48, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher host rna
    Evaluation of the coextraction method (Method 1) (A) Image of the electrophoresis gel for three independent <t>RNA</t> samples extracted from mice infected with M. ulcerans . L, RNA ladder. (B) Electropherogram: example of sample RNA derived from lane 1. (C) Table with RNA concentrations (ng/μl), indicator of purity (ratio A260/A280), integrity of RNA samples (RQI, RNA Quality Indicator), and total quantity (μg) of RNA extracted with Method 1. (D) Electropherogram: example of sample RNA before and after <t>MicrobEnrich</t> (ME) treatment. The 18S and 28S (mouse), and 16S and 23S (bacterial) rRNA bands are indicated in red and green, respectively.
    Host Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna levels total rna
    Evaluation of the coextraction method (Method 1) (A) Image of the electrophoresis gel for three independent <t>RNA</t> samples extracted from mice infected with M. ulcerans . L, RNA ladder. (B) Electropherogram: example of sample RNA derived from lane 1. (C) Table with RNA concentrations (ng/μl), indicator of purity (ratio A260/A280), integrity of RNA samples (RQI, RNA Quality Indicator), and total quantity (μg) of RNA extracted with Method 1. (D) Electropherogram: example of sample RNA before and after <t>MicrobEnrich</t> (ME) treatment. The 18S and 28S (mouse), and 16S and 23S (bacterial) rRNA bands are indicated in red and green, respectively.
    Rna Levels Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hcv rna levels
    The inhibitory effect of LPL on HCVcc infection is only partly related to its catalytic activity. (A) Huh7.5 cells were pre-incubated with 1 µg/ml LPL at 4°C in the presence or absence of 50 µg/ml THL before infection with JFH1. The infected cells were grown for 24 h and <t>HCV</t> <t>RNA</t> was then extracted and quantified by RT-qPCR. (B) THL does not influence HCV replication. Huh7.5 cells were pre-incubated with indicated concentrations of THL before cell infection with JFH-1, as for experiments with LPL. THL was maintained in the medium for 24 h post infection. HCV RNA was then extracted and quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL and THL.
    Hcv Rna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna endogenous control
    The inhibitory effect of LPL on HCVcc infection is only partly related to its catalytic activity. (A) Huh7.5 cells were pre-incubated with 1 µg/ml LPL at 4°C in the presence or absence of 50 µg/ml THL before infection with JFH1. The infected cells were grown for 24 h and <t>HCV</t> <t>RNA</t> was then extracted and quantified by RT-qPCR. (B) THL does not influence HCV replication. Huh7.5 cells were pre-incubated with indicated concentrations of THL before cell infection with JFH-1, as for experiments with LPL. THL was maintained in the medium for 24 h post infection. HCV RNA was then extracted and quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL and THL.
    Rna Endogenous Control, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher esophagus rna
    The inhibitory effect of LPL on HCVcc infection is only partly related to its catalytic activity. (A) Huh7.5 cells were pre-incubated with 1 µg/ml LPL at 4°C in the presence or absence of 50 µg/ml THL before infection with JFH1. The infected cells were grown for 24 h and <t>HCV</t> <t>RNA</t> was then extracted and quantified by RT-qPCR. (B) THL does not influence HCV replication. Huh7.5 cells were pre-incubated with indicated concentrations of THL before cell infection with JFH-1, as for experiments with LPL. THL was maintained in the medium for 24 h post infection. HCV RNA was then extracted and quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL and THL.
    Esophagus Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher spike rna levels
    The inhibitory effect of LPL on HCVcc infection is only partly related to its catalytic activity. (A) Huh7.5 cells were pre-incubated with 1 µg/ml LPL at 4°C in the presence or absence of 50 µg/ml THL before infection with JFH1. The infected cells were grown for 24 h and <t>HCV</t> <t>RNA</t> was then extracted and quantified by RT-qPCR. (B) THL does not influence HCV replication. Huh7.5 cells were pre-incubated with indicated concentrations of THL before cell infection with JFH-1, as for experiments with LPL. THL was maintained in the medium for 24 h post infection. HCV RNA was then extracted and quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL and THL.
    Spike Rna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gapdh rna
    The inhibitory effect of LPL on HCVcc infection is only partly related to its catalytic activity. (A) Huh7.5 cells were pre-incubated with 1 µg/ml LPL at 4°C in the presence or absence of 50 µg/ml THL before infection with JFH1. The infected cells were grown for 24 h and <t>HCV</t> <t>RNA</t> was then extracted and quantified by RT-qPCR. (B) THL does not influence HCV replication. Huh7.5 cells were pre-incubated with indicated concentrations of THL before cell infection with JFH-1, as for experiments with LPL. THL was maintained in the medium for 24 h post infection. HCV RNA was then extracted and quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL and THL.
    Gapdh Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqman rna to ct 1 step kit
    The inhibitory effect of LPL on HCVcc infection is only partly related to its catalytic activity. (A) Huh7.5 cells were pre-incubated with 1 µg/ml LPL at 4°C in the presence or absence of 50 µg/ml THL before infection with JFH1. The infected cells were grown for 24 h and <t>HCV</t> <t>RNA</t> was then extracted and quantified by RT-qPCR. (B) THL does not influence HCV replication. Huh7.5 cells were pre-incubated with indicated concentrations of THL before cell infection with JFH-1, as for experiments with LPL. THL was maintained in the medium for 24 h post infection. HCV RNA was then extracted and quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL and THL.
    Taqman Rna To Ct 1 Step Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2636 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher endogenous 18s rna levels
    The inhibitory effect of LPL on HCVcc infection is only partly related to its catalytic activity. (A) Huh7.5 cells were pre-incubated with 1 µg/ml LPL at 4°C in the presence or absence of 50 µg/ml THL before infection with JFH1. The infected cells were grown for 24 h and <t>HCV</t> <t>RNA</t> was then extracted and quantified by RT-qPCR. (B) THL does not influence HCV replication. Huh7.5 cells were pre-incubated with indicated concentrations of THL before cell infection with JFH-1, as for experiments with LPL. THL was maintained in the medium for 24 h post infection. HCV RNA was then extracted and quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL and THL.
    Endogenous 18s Rna Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Serum shock is sufficient to synchronize circadian gene expression in C2C12 myotubes. Stable clones of C2C12 cells (CE-4.7 MyoD ) were differentiated for 5 days prior to serum shock. RNA was isolated at the indicated time post-serum shock and used to determine the expression level of Bmal1 and MyoD by RT-PCR. The target transcript levels were normalized to Rpl26 transcript levels and then normalized to the highest point across all time points. The data for the graphs represent the mean values ± SEM for three independent clones for the reporter gene. The expression of endogenous Bmal1 ( A ), endogenous MyoD ( B ) all displayed a circadian oscillation as indicated by significant differences in peak and trough expression. For each respective gene, one-way ANOVA followed by Tukey post-hoc test identified significant difference ( P

    Journal: Nucleic Acids Research

    Article Title: A non-canonical E-box within the MyoD core enhancer is necessary for circadian expression in skeletal muscle

    doi: 10.1093/nar/gkr1297

    Figure Lengend Snippet: Serum shock is sufficient to synchronize circadian gene expression in C2C12 myotubes. Stable clones of C2C12 cells (CE-4.7 MyoD ) were differentiated for 5 days prior to serum shock. RNA was isolated at the indicated time post-serum shock and used to determine the expression level of Bmal1 and MyoD by RT-PCR. The target transcript levels were normalized to Rpl26 transcript levels and then normalized to the highest point across all time points. The data for the graphs represent the mean values ± SEM for three independent clones for the reporter gene. The expression of endogenous Bmal1 ( A ), endogenous MyoD ( B ) all displayed a circadian oscillation as indicated by significant differences in peak and trough expression. For each respective gene, one-way ANOVA followed by Tukey post-hoc test identified significant difference ( P

    Article Snippet: Real-time PCR To analyze gene expression levels, 1 µg of RNA was reverse-transcribed to cDNA using Superscript III First Strand Synthesis system (Invitrogen).

    Techniques: Expressing, Clone Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    Nucleotide biases seen at ligation sites and varies between individual miRNAs. ( A ) SeqLogo representation of the base composition of the degenerate Ns over all miRNAs and select miRs representing high (miR-21) mid (Let-7i, miR-96) and low (miR-151) expression. The height of the letter representing the base is proportional to its probability. Bases 1–4 are at the 3′ end of 5′ adapter. Bases 5–8 are at the 5′ end of 3′ adapter. Bases 9–12 follow barcode in 3′ adapter. ( B ) Cumulative divergence from expected probability of nucleotide composition at each base across all miRNAs, and the same select miRs as ( A ). Data shown is from a 500 ng cellular RNA input sample.

    Journal: Scientific Reports

    Article Title: High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing

    doi: 10.1038/s41598-018-38458-7

    Figure Lengend Snippet: Nucleotide biases seen at ligation sites and varies between individual miRNAs. ( A ) SeqLogo representation of the base composition of the degenerate Ns over all miRNAs and select miRs representing high (miR-21) mid (Let-7i, miR-96) and low (miR-151) expression. The height of the letter representing the base is proportional to its probability. Bases 1–4 are at the 3′ end of 5′ adapter. Bases 5–8 are at the 5′ end of 3′ adapter. Bases 9–12 follow barcode in 3′ adapter. ( B ) Cumulative divergence from expected probability of nucleotide composition at each base across all miRNAs, and the same select miRs as ( A ). Data shown is from a 500 ng cellular RNA input sample.

    Article Snippet: With the optimized method presented here, miRNA libraries were successfully prepared from plasma samples with RNA levels undetectable by the Thermo Scientific Nanodrop One ( < 1.6 ng/μL) or Agilent Bioanalyzer RNA 6000 Pico kit ( < 50 pg/μL).

    Techniques: Ligation, Expressing

    Unique molecular identifiers (UMIs) collapse duplicate reads and reveal linear relationship at low PCR cycles between total and collapsed counts, but drop out at high PCR cycles. ( A-C ) Effect of increasing length of UMI on number of miRNA counts following collapsing of miRNA + UMI, compared to total “raw” count. Number to left of + sign represents Ns on the 5′ adapter while numbers to right represent 3′ adapter. Insert represent collapse on miRNAs alone (i.e. without and UMI). Raw represents uncollapsed read count. Analysis shown for all miRNAs (A), a highly expressed miRNA (miR-21, B) and intermediately expressed miRNA (miR-96, C ). ( D ) Correlation plot of log10 counts per million for each miRNA comparing collapsed versus uncollapsed (total) reads for a library amplified for 14 cycles. All 12 random nucleotides were used for collapsing. ( E ) Same as D, but amplified for 24 cycles showing reduction in correlation for low expressed miRNAs. ( F ) Same as D, but comparing only collapsed reads between library amplified for 14 versus 24 cycles, showing much poorer correlation for low to intermediate expressed miRNAs. ( G-I ) Direct comparison of read counts for each miRNA from libraries differing in the number of PCR amplification cycles. miRNAs are ordered from high to low expression in 14 cycle library. ( G ) Uncollapsed (total) counts per million. ( H ) Collapsed counts per million. ( I ) Percent unique reads (i.e. collapsed counts/total counts *100). Note noise created by high PCR cycle number on the lowly to intermediately expressed miRNAs. All libraries were made from one cellular input RNA.

    Journal: Scientific Reports

    Article Title: High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing

    doi: 10.1038/s41598-018-38458-7

    Figure Lengend Snippet: Unique molecular identifiers (UMIs) collapse duplicate reads and reveal linear relationship at low PCR cycles between total and collapsed counts, but drop out at high PCR cycles. ( A-C ) Effect of increasing length of UMI on number of miRNA counts following collapsing of miRNA + UMI, compared to total “raw” count. Number to left of + sign represents Ns on the 5′ adapter while numbers to right represent 3′ adapter. Insert represent collapse on miRNAs alone (i.e. without and UMI). Raw represents uncollapsed read count. Analysis shown for all miRNAs (A), a highly expressed miRNA (miR-21, B) and intermediately expressed miRNA (miR-96, C ). ( D ) Correlation plot of log10 counts per million for each miRNA comparing collapsed versus uncollapsed (total) reads for a library amplified for 14 cycles. All 12 random nucleotides were used for collapsing. ( E ) Same as D, but amplified for 24 cycles showing reduction in correlation for low expressed miRNAs. ( F ) Same as D, but comparing only collapsed reads between library amplified for 14 versus 24 cycles, showing much poorer correlation for low to intermediate expressed miRNAs. ( G-I ) Direct comparison of read counts for each miRNA from libraries differing in the number of PCR amplification cycles. miRNAs are ordered from high to low expression in 14 cycle library. ( G ) Uncollapsed (total) counts per million. ( H ) Collapsed counts per million. ( I ) Percent unique reads (i.e. collapsed counts/total counts *100). Note noise created by high PCR cycle number on the lowly to intermediately expressed miRNAs. All libraries were made from one cellular input RNA.

    Article Snippet: With the optimized method presented here, miRNA libraries were successfully prepared from plasma samples with RNA levels undetectable by the Thermo Scientific Nanodrop One ( < 1.6 ng/μL) or Agilent Bioanalyzer RNA 6000 Pico kit ( < 50 pg/μL).

    Techniques: Polymerase Chain Reaction, Amplification, Expressing

    Modifications to high-throughput sequencing method improves capture of miRNAs. ( A ) Schematic of protocol to prepare miRNA libraries for sequencing. Modifications from original protocol noted in bold. ( B ) Percentage of different classes of RNAs captured from a plasma sample using the original conditions (0.85 μM 3′ adapter, 3.3 μM unmodified 5′ adapter). Ligations were performed in triplicate from the same RNA. Each replicate is shown as an individual bar. Note the low percentage of reads mapping to miRNAs (red). ( C ) Percentage of mature miRNAs captured using the optimized conditions (0.05 μΜ 3′ adapter, 0.33 μΜ amino-modified 5′ adapter) were compared to original conditions in three independent biological samples. The ligation reactions were performed in triplicate for each sample and protocol. Each replicate is shown as a black dot. Red dot represents average. ( D ) The average percentage of reads mapping to different classes of RNA for each sample and condition shown in C.

    Journal: Scientific Reports

    Article Title: High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing

    doi: 10.1038/s41598-018-38458-7

    Figure Lengend Snippet: Modifications to high-throughput sequencing method improves capture of miRNAs. ( A ) Schematic of protocol to prepare miRNA libraries for sequencing. Modifications from original protocol noted in bold. ( B ) Percentage of different classes of RNAs captured from a plasma sample using the original conditions (0.85 μM 3′ adapter, 3.3 μM unmodified 5′ adapter). Ligations were performed in triplicate from the same RNA. Each replicate is shown as an individual bar. Note the low percentage of reads mapping to miRNAs (red). ( C ) Percentage of mature miRNAs captured using the optimized conditions (0.05 μΜ 3′ adapter, 0.33 μΜ amino-modified 5′ adapter) were compared to original conditions in three independent biological samples. The ligation reactions were performed in triplicate for each sample and protocol. Each replicate is shown as a black dot. Red dot represents average. ( D ) The average percentage of reads mapping to different classes of RNA for each sample and condition shown in C.

    Article Snippet: With the optimized method presented here, miRNA libraries were successfully prepared from plasma samples with RNA levels undetectable by the Thermo Scientific Nanodrop One ( < 1.6 ng/μL) or Agilent Bioanalyzer RNA 6000 Pico kit ( < 50 pg/μL).

    Techniques: Next-Generation Sequencing, Sequencing, Modification, Ligation

    Improved miRNA capture also seen at low concentrations of input RNA. ( A ) The percentage of reads mapping to miRNAs at the indicated input following either the optimized (0.05 μΜ 3′ adapter, 0.33 μΜ amino-modified 5′adapter) or the original (0.85 μM 3′ adapter, 3.3 μM unmodified 5′ adapter) protocol. Black dots represent the three replicates from each input RNA for each sample, protocol, and concentration. Red dots represent average. ( B ) The average percentage of reads mapping to different RNA classes for each sample, protocol, and concentration.

    Journal: Scientific Reports

    Article Title: High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing

    doi: 10.1038/s41598-018-38458-7

    Figure Lengend Snippet: Improved miRNA capture also seen at low concentrations of input RNA. ( A ) The percentage of reads mapping to miRNAs at the indicated input following either the optimized (0.05 μΜ 3′ adapter, 0.33 μΜ amino-modified 5′adapter) or the original (0.85 μM 3′ adapter, 3.3 μM unmodified 5′ adapter) protocol. Black dots represent the three replicates from each input RNA for each sample, protocol, and concentration. Red dots represent average. ( B ) The average percentage of reads mapping to different RNA classes for each sample, protocol, and concentration.

    Article Snippet: With the optimized method presented here, miRNA libraries were successfully prepared from plasma samples with RNA levels undetectable by the Thermo Scientific Nanodrop One ( < 1.6 ng/μL) or Agilent Bioanalyzer RNA 6000 Pico kit ( < 50 pg/μL).

    Techniques: Modification, Concentration Assay

    Evaluation of the coextraction method (Method 1) (A) Image of the electrophoresis gel for three independent RNA samples extracted from mice infected with M. ulcerans . L, RNA ladder. (B) Electropherogram: example of sample RNA derived from lane 1. (C) Table with RNA concentrations (ng/μl), indicator of purity (ratio A260/A280), integrity of RNA samples (RQI, RNA Quality Indicator), and total quantity (μg) of RNA extracted with Method 1. (D) Electropherogram: example of sample RNA before and after MicrobEnrich (ME) treatment. The 18S and 28S (mouse), and 16S and 23S (bacterial) rRNA bands are indicated in red and green, respectively.

    Journal: Frontiers in Microbiology

    Article Title: An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans

    doi: 10.3389/fmicb.2017.00512

    Figure Lengend Snippet: Evaluation of the coextraction method (Method 1) (A) Image of the electrophoresis gel for three independent RNA samples extracted from mice infected with M. ulcerans . L, RNA ladder. (B) Electropherogram: example of sample RNA derived from lane 1. (C) Table with RNA concentrations (ng/μl), indicator of purity (ratio A260/A280), integrity of RNA samples (RQI, RNA Quality Indicator), and total quantity (μg) of RNA extracted with Method 1. (D) Electropherogram: example of sample RNA before and after MicrobEnrich (ME) treatment. The 18S and 28S (mouse), and 16S and 23S (bacterial) rRNA bands are indicated in red and green, respectively.

    Article Snippet: Total RNA was purified with the RNeasy Midi kit (Qiagen), with DNase treatment, according to the manufacturer’s protocol, and eluted in 100 μl RNase- and DNase-free water. (iii) DNase treatment: Contaminating DNA was removed by retreating the RNA with DNase, for 45 min at 37°C, with the TURBO DNA-free kit (Ambion), according to the manufacturer’s protocol. (iv) Eukaryotic RNA removal: To reduce the levels of contaminating host RNA from the samples, MICROBEnrich kit (Ambion) was used according to the manufacturer’s protocol.

    Techniques: Electrophoresis, Mouse Assay, Infection, Derivative Assay

    Percentage of reads aligned with the M. ulcerans and mouse genomes. M1, Total RNA sample obtained with Method 1; M1_ME, Total RNA sample obtained with Method 1 after enrichment with MICROBEnrich (ME); M2, Bacterial RNA-enriched sample obtained by differential lysis (Method 2). M2-1, M2-2, M2-3 represent three replicates for Method 2.

    Journal: Frontiers in Microbiology

    Article Title: An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans

    doi: 10.3389/fmicb.2017.00512

    Figure Lengend Snippet: Percentage of reads aligned with the M. ulcerans and mouse genomes. M1, Total RNA sample obtained with Method 1; M1_ME, Total RNA sample obtained with Method 1 after enrichment with MICROBEnrich (ME); M2, Bacterial RNA-enriched sample obtained by differential lysis (Method 2). M2-1, M2-2, M2-3 represent three replicates for Method 2.

    Article Snippet: Total RNA was purified with the RNeasy Midi kit (Qiagen), with DNase treatment, according to the manufacturer’s protocol, and eluted in 100 μl RNase- and DNase-free water. (iii) DNase treatment: Contaminating DNA was removed by retreating the RNA with DNase, for 45 min at 37°C, with the TURBO DNA-free kit (Ambion), according to the manufacturer’s protocol. (iv) Eukaryotic RNA removal: To reduce the levels of contaminating host RNA from the samples, MICROBEnrich kit (Ambion) was used according to the manufacturer’s protocol.

    Techniques: Lysis

    The inhibitory effect of LPL on HCVcc infection is only partly related to its catalytic activity. (A) Huh7.5 cells were pre-incubated with 1 µg/ml LPL at 4°C in the presence or absence of 50 µg/ml THL before infection with JFH1. The infected cells were grown for 24 h and HCV RNA was then extracted and quantified by RT-qPCR. (B) THL does not influence HCV replication. Huh7.5 cells were pre-incubated with indicated concentrations of THL before cell infection with JFH-1, as for experiments with LPL. THL was maintained in the medium for 24 h post infection. HCV RNA was then extracted and quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL and THL.

    Journal: PLoS ONE

    Article Title: Lipoprotein Lipase Inhibits Hepatitis C Virus (HCV) Infection by Blocking Virus Cell Entry

    doi: 10.1371/journal.pone.0026637

    Figure Lengend Snippet: The inhibitory effect of LPL on HCVcc infection is only partly related to its catalytic activity. (A) Huh7.5 cells were pre-incubated with 1 µg/ml LPL at 4°C in the presence or absence of 50 µg/ml THL before infection with JFH1. The infected cells were grown for 24 h and HCV RNA was then extracted and quantified by RT-qPCR. (B) THL does not influence HCV replication. Huh7.5 cells were pre-incubated with indicated concentrations of THL before cell infection with JFH-1, as for experiments with LPL. THL was maintained in the medium for 24 h post infection. HCV RNA was then extracted and quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL and THL.

    Article Snippet: HCV RNA levels are expressed in IU relative to a HCV RNA quantification panel from Acrometrix (Berkeley, CA, USA).

    Techniques: Infection, Activity Assay, Incubation, Quantitative RT-PCR

    LPL inhibits cell infection by the JFH-1 and J6/JFH-1 strains produced in vitro and in vivo in a chimeric uPA-SCID mouse model. The HCVcc strains JFH-1 (A) and J6/JFH-1 (B) were produced in the Huh7.5 hepatoma cell line. Cells were incubated with (or without) LPL for 30 min at 4°C and then with virus preparations for 2 h at 37°C to allow infection. RNA was extracted from cells 24 h post infection and HCV RNA was quantified by RT-qPCR. The data obtained were normalized with respect to levels of GADPH. The mJFH-1 (A) and mJ6/JFH-1 (B) correspond to HCVcc strains produced in chimeric uPA-SCID mice into which we transplanted human hepatocytes. Serum samples collected from infected mice were pooled and their capacity to infect Huh7.5 cells was assessed in the presence and absence of LPL, as outlined above. Cells infected in the absence (black bar) and in the presence of LPL (gray bar). The data are expressed as the amount of HCV RNA detected in cells infected in the presence of LPL as compared with the amount of HCV RNA in cells infected in the absence of LPL, expressed as a percentage.

    Journal: PLoS ONE

    Article Title: Lipoprotein Lipase Inhibits Hepatitis C Virus (HCV) Infection by Blocking Virus Cell Entry

    doi: 10.1371/journal.pone.0026637

    Figure Lengend Snippet: LPL inhibits cell infection by the JFH-1 and J6/JFH-1 strains produced in vitro and in vivo in a chimeric uPA-SCID mouse model. The HCVcc strains JFH-1 (A) and J6/JFH-1 (B) were produced in the Huh7.5 hepatoma cell line. Cells were incubated with (or without) LPL for 30 min at 4°C and then with virus preparations for 2 h at 37°C to allow infection. RNA was extracted from cells 24 h post infection and HCV RNA was quantified by RT-qPCR. The data obtained were normalized with respect to levels of GADPH. The mJFH-1 (A) and mJ6/JFH-1 (B) correspond to HCVcc strains produced in chimeric uPA-SCID mice into which we transplanted human hepatocytes. Serum samples collected from infected mice were pooled and their capacity to infect Huh7.5 cells was assessed in the presence and absence of LPL, as outlined above. Cells infected in the absence (black bar) and in the presence of LPL (gray bar). The data are expressed as the amount of HCV RNA detected in cells infected in the presence of LPL as compared with the amount of HCV RNA in cells infected in the absence of LPL, expressed as a percentage.

    Article Snippet: HCV RNA levels are expressed in IU relative to a HCV RNA quantification panel from Acrometrix (Berkeley, CA, USA).

    Techniques: Infection, Produced, In Vitro, In Vivo, Incubation, Quantitative RT-PCR, Mouse Assay

    LPL affects HCV attachment and early stages of the virus cell cycle. (A) Effect of LPL on virus attachment to Huh7.5 cells. Huh7.5 cells were pre-incubated with various concentrations of LPL (1–9 µg/ml) for 30 min at 4°C. An aliquot of cell culture supernatant containing JFH-1 was incubated with LPL-pretreated Huh7.5 cells for 30 min at 4°C. The cells were washed and the RNA associated with them was extracted. HCV RNA was quantified by RT-qPCR. (B) Effect of LPL on early steps of HCV infection. JFH-1 was first adsorbed onto Huh7.5 cells by incubation for 45 min at 4°C. Cells were washed with cold medium to remove any unbound virus. Complete medium, warmed to 37°C, was then added and incubated with the cells at 37°C. LPL was added to a concentration of 1 µg/ml at various time points (0, 5, 10, 15 or 20 min) after the transfer of cells to 37°C, with or without the addition of 50 µg/ml THL to block its enzymatic activity. Cells were grown for 24 h. RNA was then extracted and HCV RNA was quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL.

    Journal: PLoS ONE

    Article Title: Lipoprotein Lipase Inhibits Hepatitis C Virus (HCV) Infection by Blocking Virus Cell Entry

    doi: 10.1371/journal.pone.0026637

    Figure Lengend Snippet: LPL affects HCV attachment and early stages of the virus cell cycle. (A) Effect of LPL on virus attachment to Huh7.5 cells. Huh7.5 cells were pre-incubated with various concentrations of LPL (1–9 µg/ml) for 30 min at 4°C. An aliquot of cell culture supernatant containing JFH-1 was incubated with LPL-pretreated Huh7.5 cells for 30 min at 4°C. The cells were washed and the RNA associated with them was extracted. HCV RNA was quantified by RT-qPCR. (B) Effect of LPL on early steps of HCV infection. JFH-1 was first adsorbed onto Huh7.5 cells by incubation for 45 min at 4°C. Cells were washed with cold medium to remove any unbound virus. Complete medium, warmed to 37°C, was then added and incubated with the cells at 37°C. LPL was added to a concentration of 1 µg/ml at various time points (0, 5, 10, 15 or 20 min) after the transfer of cells to 37°C, with or without the addition of 50 µg/ml THL to block its enzymatic activity. Cells were grown for 24 h. RNA was then extracted and HCV RNA was quantified by RT-qPCR. Results are expressed as a percent of RNA as compared with control cells infected in the absence of LPL.

    Article Snippet: HCV RNA levels are expressed in IU relative to a HCV RNA quantification panel from Acrometrix (Berkeley, CA, USA).

    Techniques: Incubation, Cell Culture, Quantitative RT-PCR, Infection, Concentration Assay, Blocking Assay, Activity Assay

    Cell infection with low- and high-density virus populations from iodixanol gradients in the presence or absence of LPL. The pooled peak fractions of the low- and high-density virus populations obtained after centrifugation through iodixanol gradients of JFH1 grown in cell culture (A) and serum samples from the m-JFH1 chimeric mouse model (B) (both gradients are shown in Figure 2 ) were used to infect cells in the presence or absence of 1 µg/ml LPL, as described in the Materials and Methods section. Cells were grown for 48 h at 37°C and HCV RNA was extracted and quantified by RT-qPCR. The results were normalized with respect to the cellular gene GAPDH, with the GAPDH Control Kit. The data are expressed as the amount of HCV RNA detected in cells infected with the pooled fractions from the two major virus populations in the presence of LPL as compared with the amount of HCV RNA in cells infected with the same fractions in the absence of LPL, expressed as a percentage.

    Journal: PLoS ONE

    Article Title: Lipoprotein Lipase Inhibits Hepatitis C Virus (HCV) Infection by Blocking Virus Cell Entry

    doi: 10.1371/journal.pone.0026637

    Figure Lengend Snippet: Cell infection with low- and high-density virus populations from iodixanol gradients in the presence or absence of LPL. The pooled peak fractions of the low- and high-density virus populations obtained after centrifugation through iodixanol gradients of JFH1 grown in cell culture (A) and serum samples from the m-JFH1 chimeric mouse model (B) (both gradients are shown in Figure 2 ) were used to infect cells in the presence or absence of 1 µg/ml LPL, as described in the Materials and Methods section. Cells were grown for 48 h at 37°C and HCV RNA was extracted and quantified by RT-qPCR. The results were normalized with respect to the cellular gene GAPDH, with the GAPDH Control Kit. The data are expressed as the amount of HCV RNA detected in cells infected with the pooled fractions from the two major virus populations in the presence of LPL as compared with the amount of HCV RNA in cells infected with the same fractions in the absence of LPL, expressed as a percentage.

    Article Snippet: HCV RNA levels are expressed in IU relative to a HCV RNA quantification panel from Acrometrix (Berkeley, CA, USA).

    Techniques: Infection, Centrifugation, Cell Culture, Quantitative RT-PCR

    Iodixanol gradient analysis of the JFH-1 and J6/JFH-1 strains produced in vitro and in vivo . The supernatants from infected Huh7.5 cells producing JFH-1 (JFH-1, shown in A and B) and J6/JFH-1 (shown in E) were subjected to isopycnic centrifugation through iodixanol gradients, as described in Materials and Methods . Pooled serum samples from the chimeric uPA-SCID mice were also subjected to centrifugation on the same type of gradient. Representative profiles are shown in C and D for mice inoculated with JFH-1 (mJFH-1) and in F for mice inoculated with J6/JFH-1 (mJ6/JFH-1). HCV core antigen in gradient fractions was quantified by ELISA, HCV RNA was quantified by RT-qPCR, and ApoB and cholesterol were determined by ELISA. Infectivity for fractionated J6/JFH-1 (representative for both strains) grown in Huh7.5 cells is shown in E and that for the corresponding mouse serum (mJ6/JFH-1) is shown in F. The fractions (25 µl) were used to infect Huh7.5 cells. Cells were incubated for 48 h at 37°C; total RNA was then extracted and HCV-RNA levels were quantified by RT-qPCR. The results were normalized, taking into account the initial HCV-RNA content in each sample analyzed, as determined by RT-qPCR, and are expressed as a ratio of these two values.

    Journal: PLoS ONE

    Article Title: Lipoprotein Lipase Inhibits Hepatitis C Virus (HCV) Infection by Blocking Virus Cell Entry

    doi: 10.1371/journal.pone.0026637

    Figure Lengend Snippet: Iodixanol gradient analysis of the JFH-1 and J6/JFH-1 strains produced in vitro and in vivo . The supernatants from infected Huh7.5 cells producing JFH-1 (JFH-1, shown in A and B) and J6/JFH-1 (shown in E) were subjected to isopycnic centrifugation through iodixanol gradients, as described in Materials and Methods . Pooled serum samples from the chimeric uPA-SCID mice were also subjected to centrifugation on the same type of gradient. Representative profiles are shown in C and D for mice inoculated with JFH-1 (mJFH-1) and in F for mice inoculated with J6/JFH-1 (mJ6/JFH-1). HCV core antigen in gradient fractions was quantified by ELISA, HCV RNA was quantified by RT-qPCR, and ApoB and cholesterol were determined by ELISA. Infectivity for fractionated J6/JFH-1 (representative for both strains) grown in Huh7.5 cells is shown in E and that for the corresponding mouse serum (mJ6/JFH-1) is shown in F. The fractions (25 µl) were used to infect Huh7.5 cells. Cells were incubated for 48 h at 37°C; total RNA was then extracted and HCV-RNA levels were quantified by RT-qPCR. The results were normalized, taking into account the initial HCV-RNA content in each sample analyzed, as determined by RT-qPCR, and are expressed as a ratio of these two values.

    Article Snippet: HCV RNA levels are expressed in IU relative to a HCV RNA quantification panel from Acrometrix (Berkeley, CA, USA).

    Techniques: Produced, In Vitro, In Vivo, Infection, Centrifugation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Incubation