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  • 99
    Roche pcr amplification
    <t>PCR</t> results of <t>mecA</t> gene in 7 isolates staphylococcus epidermidis. Column 10: Size marker (50 bp). Column 8, 1-6: Isolates of Staphylococcus epidermidis containing mecA gene. Column 7: Positive control of staphylococcus aureus ATC 33591. Column 9: Negative control of staphylococcus aureus ATCC 25923
    Pcr Amplification, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 9762 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche 1x polymerase chain reaction pcr buffer
    <t>PCR</t> results of <t>mecA</t> gene in 7 isolates staphylococcus epidermidis. Column 10: Size marker (50 bp). Column 8, 1-6: Isolates of Staphylococcus epidermidis containing mecA gene. Column 7: Positive control of staphylococcus aureus ATC 33591. Column 9: Negative control of staphylococcus aureus ATCC 25923
    1x Polymerase Chain Reaction Pcr Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche high fidelity pcr
    Detection of SARS CoV from 18 subjects by real-time quantitative <t>PCR.</t> Data for <t>RNA</t> from 18 subjects are shown. The viral load is indicated as number of copies per ml of blood. Subject 1, 2,020 copies; subject 2, 9,100 copies; subject 3, 3,120 copies; subject 4, 9,600 copies. A sample would be considered positive if it generated a typical amplification curve within the 45 cycles. Negative signals from subjects 5 to 18 and the nontemplate control (water) are shown. The inset graph shows melting-curve analysis of the PCR products from the 18 subjects. The x axis denotes the temperature ( o C), and the y axis denotes the fluorescence intensity (F2) over the background level.
    High Fidelity Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 8731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche transcription polymerase chain reaction rt pcr
    Hypoxic regulation of VEGF by K-ras. A , Relative <t>mRNA</t> levels of VEGF, as evaluated by quantitative <t>RT-PCR,</t> in Caco2 cells transfected with a K-ras-specific siRNA construct or a non targeting control and exposed to normoxia or hypoxia. The data are expressed as fold change as compared to siRNA control cells in normoxia, normalized to 1. Columns, average of at least three experiments; bars, SEM. *, P
    Transcription Polymerase Chain Reaction Rt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche high fidelity pcr kit
    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in <t>Igκ</t> wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative <t>PCR</t> via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.
    High Fidelity Pcr Kit, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Roche fluorescence ratio polymerase chain reaction pcr instrument
    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in <t>Igκ</t> wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative <t>PCR</t> via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.
    Fluorescence Ratio Polymerase Chain Reaction Pcr Instrument, supplied by Roche, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche expand high fidelity polymerase chain reaction pcr system
    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in <t>Igκ</t> wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative <t>PCR</t> via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.
    Expand High Fidelity Polymerase Chain Reaction Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche polymerase chain reaction pcr based amplicor hpv test
    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in <t>Igκ</t> wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative <t>PCR</t> via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.
    Polymerase Chain Reaction Pcr Based Amplicor Hpv Test, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche extended polymerase chain reaction pcr
    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in <t>Igκ</t> wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative <t>PCR</t> via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.
    Extended Polymerase Chain Reaction Pcr, supplied by Roche, 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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    90
    Roche polymerase chain reaction pcr amplified
    Heat shock and cadmium sulfate change the alternative splicing pattern of the adenovirus E1A minigene. (A) Schematic representation of the alternative spliced isoforms of E1A pre-mRNA. The major isoforms 9S, 12S, and 13S are generated by alternative selection of the 5′ splice sites. The minor isoforms 10S and 11S involve the usage of additional internal 3′ splice sites. Sizes, in base pairs, of the corresponding <t>RT-PCR</t> products obtained with the use of the E1A-569 and E1A-1315 primers are shown. (B) In vivo alternative splicing of E1A pre-mRNA is affected by heat shock and by cadmium sulfate. The pCMVE1A plasmid containing the E1A minigene was transfected in HeLa cells. Twenty-four hours after transfection total RNAs were extracted from unstressed cells (37°C) or from cells heat-shocked for 1 h at 42°C. RNAs were also prepared from cells allowed to recover 1, 2, 4, or 6 h at 37°C after heat shock. RNAs were also extracted from cells treated for 3 or 5 h with 30 μM of cadmium sulfate, another induced of <t>HAP</t> bodies. RNA were analyzed by RT-PCR with E1A-569 and E1A-1315 primers. RT-PCR products were resolved on 5% polyacrylamide gel and detected by autoradiography. (C) ImageQuant PhosphorImager quantification of the major E1A isoforms: white bars, light gray bars, and dark gray bars, represent 13S, 12S, and 9S molecules, respectively. Percentages of the three isoforms are the average of at least three independent experiments. Error bars are shown.
    Polymerase Chain Reaction Pcr Amplified, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche polymerase chain reaction pcr fragment
    Heat shock and cadmium sulfate change the alternative splicing pattern of the adenovirus E1A minigene. (A) Schematic representation of the alternative spliced isoforms of E1A pre-mRNA. The major isoforms 9S, 12S, and 13S are generated by alternative selection of the 5′ splice sites. The minor isoforms 10S and 11S involve the usage of additional internal 3′ splice sites. Sizes, in base pairs, of the corresponding <t>RT-PCR</t> products obtained with the use of the E1A-569 and E1A-1315 primers are shown. (B) In vivo alternative splicing of E1A pre-mRNA is affected by heat shock and by cadmium sulfate. The pCMVE1A plasmid containing the E1A minigene was transfected in HeLa cells. Twenty-four hours after transfection total RNAs were extracted from unstressed cells (37°C) or from cells heat-shocked for 1 h at 42°C. RNAs were also prepared from cells allowed to recover 1, 2, 4, or 6 h at 37°C after heat shock. RNAs were also extracted from cells treated for 3 or 5 h with 30 μM of cadmium sulfate, another induced of <t>HAP</t> bodies. RNA were analyzed by RT-PCR with E1A-569 and E1A-1315 primers. RT-PCR products were resolved on 5% polyacrylamide gel and detected by autoradiography. (C) ImageQuant PhosphorImager quantification of the major E1A isoforms: white bars, light gray bars, and dark gray bars, represent 13S, 12S, and 9S molecules, respectively. Percentages of the three isoforms are the average of at least three independent experiments. Error bars are shown.
    Polymerase Chain Reaction Pcr Fragment, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Roche emulsion pcr
    Frequency and relative expression of KIR genes in the rhesus macaque cohort . A) Y-axis indicates the percentage of animals within the cohort (n = 69) that express the indicated KIR gene. Genes not listed were not present in any animal within our cohort. Mamu-KIR2DL04 was excluded since it is not amplified by our pyrosequencing <t>amplicon.</t> B) Graph illustrates the percent of total pyrosequencing reads per animal (n = 61) for each KIR gene. Averages and SEM are represented by error bars. Each animal included had at least 100 sequencing reads. Genotyping results representing less than 1% of total reads in an animal were excluded to mitigate the influence of <t>PCR</t> artifacts. Ambiguous reads matching more than one KIR gene and splice variants were also excluded.
    Emulsion Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 2635 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche expand long template polymerase chain reaction pcr system
    Frequency and relative expression of KIR genes in the rhesus macaque cohort . A) Y-axis indicates the percentage of animals within the cohort (n = 69) that express the indicated KIR gene. Genes not listed were not present in any animal within our cohort. Mamu-KIR2DL04 was excluded since it is not amplified by our pyrosequencing <t>amplicon.</t> B) Graph illustrates the percent of total pyrosequencing reads per animal (n = 61) for each KIR gene. Averages and SEM are represented by error bars. Each animal included had at least 100 sequencing reads. Genotyping results representing less than 1% of total reads in an animal were excluded to mitigate the influence of <t>PCR</t> artifacts. Ambiguous reads matching more than one KIR gene and splice variants were also excluded.
    Expand Long Template Polymerase Chain Reaction Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche polymerase chain reaction pcr buffer
    Frequency and relative expression of KIR genes in the rhesus macaque cohort . A) Y-axis indicates the percentage of animals within the cohort (n = 69) that express the indicated KIR gene. Genes not listed were not present in any animal within our cohort. Mamu-KIR2DL04 was excluded since it is not amplified by our pyrosequencing <t>amplicon.</t> B) Graph illustrates the percent of total pyrosequencing reads per animal (n = 61) for each KIR gene. Averages and SEM are represented by error bars. Each animal included had at least 100 sequencing reads. Genotyping results representing less than 1% of total reads in an animal were excluded to mitigate the influence of <t>PCR</t> artifacts. Ambiguous reads matching more than one KIR gene and splice variants were also excluded.
    Polymerase Chain Reaction Pcr Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PCR results of mecA gene in 7 isolates staphylococcus epidermidis. Column 10: Size marker (50 bp). Column 8, 1-6: Isolates of Staphylococcus epidermidis containing mecA gene. Column 7: Positive control of staphylococcus aureus ATC 33591. Column 9: Negative control of staphylococcus aureus ATCC 25923

    Journal: Advanced Biomedical Research

    Article Title: Detection of methicillin-resistance gene in Staphylococcus epidermidis strains isolated from patients in Al-Zahra Hospital using polymerase chain reaction and minimum inhibitory concentration methods

    doi: 10.4103/2277-9175.108008

    Figure Lengend Snippet: PCR results of mecA gene in 7 isolates staphylococcus epidermidis. Column 10: Size marker (50 bp). Column 8, 1-6: Isolates of Staphylococcus epidermidis containing mecA gene. Column 7: Positive control of staphylococcus aureus ATC 33591. Column 9: Negative control of staphylococcus aureus ATCC 25923

    Article Snippet: The following primers were used for PCR amplification of mecA gene:[ ] mecA-F: 5′-TGGCTATCGTGTCACAATCG-3′ mecA-R: 5′-CTGGAACTTGTTGAGCAGAG-3′ PCR was performed in a mixture of 25 μl volume containing: 2.5 μl 10 × buffer (Roche Germany, Berlin), 0.4 μl of each dNTP (200 μm), 2.5 μl (50 mm) MgCl2, 2.5 U of Taq DNA polymerase, 10 pmol of each primer, and 5 μl of template DNA.

    Techniques: Polymerase Chain Reaction, Marker, Positive Control, Negative Control

    Genotyping results for the rs1333049 using the melting temperature shift polymerase chain reaction.

    Journal: Clinical Psychopharmacology and Neuroscience

    Article Title: Association of CDKN2B-AS1 rs1333049 with Brain Diseases: A Case-control Study and a Meta-analysis

    doi: 10.9758/cpn.2017.15.1.53

    Figure Lengend Snippet: Genotyping results for the rs1333049 using the melting temperature shift polymerase chain reaction.

    Article Snippet: The polymerase chain reaction (PCR) amplification for genotyping was performed on the Roche Light Cycler® 480 (Roche, Mannheim, Germany) according to the manufacturer’s instructions.

    Techniques: Polymerase Chain Reaction

    Detection of SARS CoV from 18 subjects by real-time quantitative PCR. Data for RNA from 18 subjects are shown. The viral load is indicated as number of copies per ml of blood. Subject 1, 2,020 copies; subject 2, 9,100 copies; subject 3, 3,120 copies; subject 4, 9,600 copies. A sample would be considered positive if it generated a typical amplification curve within the 45 cycles. Negative signals from subjects 5 to 18 and the nontemplate control (water) are shown. The inset graph shows melting-curve analysis of the PCR products from the 18 subjects. The x axis denotes the temperature ( o C), and the y axis denotes the fluorescence intensity (F2) over the background level.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection of Severe Acute Respiratory Syndrome Coronavirus in Blood of Infected Patients

    doi: 10.1128/JCM.42.1.347-350.2004

    Figure Lengend Snippet: Detection of SARS CoV from 18 subjects by real-time quantitative PCR. Data for RNA from 18 subjects are shown. The viral load is indicated as number of copies per ml of blood. Subject 1, 2,020 copies; subject 2, 9,100 copies; subject 3, 3,120 copies; subject 4, 9,600 copies. A sample would be considered positive if it generated a typical amplification curve within the 45 cycles. Negative signals from subjects 5 to 18 and the nontemplate control (water) are shown. The inset graph shows melting-curve analysis of the PCR products from the 18 subjects. The x axis denotes the temperature ( o C), and the y axis denotes the fluorescence intensity (F2) over the background level.

    Article Snippet: In the first PCR assay, 2 μl of RNA was reverse transcribed with Expand reverse transcriptase (Roche, Mannheim, Germany) using primer 5′-GGCATCATCAGAAAGAATCATCAT-3′, thereby generating 20 μl of cDNA.

    Techniques: Real-time Polymerase Chain Reaction, Generated, Amplification, Polymerase Chain Reaction, Fluorescence

    Detection of SARS CoV by real-time quantitative PCR. (A) Amplification of single-stranded RNA standards (indicated as a to e) and RNA extracted from sputum, stool, and blood spiked with virus grown in Vero E6 cells. The x axis denotes the cycle number of the quantitative PCR assay, and the y axis denotes fluorescence intensity (F2) over the background level. The nontemplate control (water) is indicated. The viral load is indicated as the number of copies per reaction: spiked sputum, 5 × 10 3 ; spiked stool, 4 × 10 3 ; spiked blood, 1 × 10 3 . RNA standards were as follows: (a) 1 × 10 6 copies per reaction, (b) 9.5 × 10 4 copies per reaction, (c) 8.7 × 10 3 copies per reaction, (d) 1.1 × 10 3 copies per reaction, and (e) 8.5 × 10 copies per reaction. The inset graph represents melting-curve analysis of the PCR products. Signals from RNA standards (a to e), spiked samples, normal samples, and nontemplate control (water) are shown. The x axis denotes the temperature ( o C), and the y axis denotes the fluorescence intensity (F2) over the background level. (B) Detection of the internal control in fluorometer channel F3 in parallel with the simultaneous amplification of the RNA standards (a to e), spiked samples, and nontemplate control is shown.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection of Severe Acute Respiratory Syndrome Coronavirus in Blood of Infected Patients

    doi: 10.1128/JCM.42.1.347-350.2004

    Figure Lengend Snippet: Detection of SARS CoV by real-time quantitative PCR. (A) Amplification of single-stranded RNA standards (indicated as a to e) and RNA extracted from sputum, stool, and blood spiked with virus grown in Vero E6 cells. The x axis denotes the cycle number of the quantitative PCR assay, and the y axis denotes fluorescence intensity (F2) over the background level. The nontemplate control (water) is indicated. The viral load is indicated as the number of copies per reaction: spiked sputum, 5 × 10 3 ; spiked stool, 4 × 10 3 ; spiked blood, 1 × 10 3 . RNA standards were as follows: (a) 1 × 10 6 copies per reaction, (b) 9.5 × 10 4 copies per reaction, (c) 8.7 × 10 3 copies per reaction, (d) 1.1 × 10 3 copies per reaction, and (e) 8.5 × 10 copies per reaction. The inset graph represents melting-curve analysis of the PCR products. Signals from RNA standards (a to e), spiked samples, normal samples, and nontemplate control (water) are shown. The x axis denotes the temperature ( o C), and the y axis denotes the fluorescence intensity (F2) over the background level. (B) Detection of the internal control in fluorometer channel F3 in parallel with the simultaneous amplification of the RNA standards (a to e), spiked samples, and nontemplate control is shown.

    Article Snippet: In the first PCR assay, 2 μl of RNA was reverse transcribed with Expand reverse transcriptase (Roche, Mannheim, Germany) using primer 5′-GGCATCATCAGAAAGAATCATCAT-3′, thereby generating 20 μl of cDNA.

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

    Hypoxic regulation of VEGF by K-ras. A , Relative mRNA levels of VEGF, as evaluated by quantitative RT-PCR, in Caco2 cells transfected with a K-ras-specific siRNA construct or a non targeting control and exposed to normoxia or hypoxia. The data are expressed as fold change as compared to siRNA control cells in normoxia, normalized to 1. Columns, average of at least three experiments; bars, SEM. *, P

    Journal: PLoS ONE

    Article Title: Hypoxia Activates the K-Ras Proto-Oncogene to Stimulate Angiogenesis and Inhibit Apoptosis in Colon Cancer Cells

    doi: 10.1371/journal.pone.0010966

    Figure Lengend Snippet: Hypoxic regulation of VEGF by K-ras. A , Relative mRNA levels of VEGF, as evaluated by quantitative RT-PCR, in Caco2 cells transfected with a K-ras-specific siRNA construct or a non targeting control and exposed to normoxia or hypoxia. The data are expressed as fold change as compared to siRNA control cells in normoxia, normalized to 1. Columns, average of at least three experiments; bars, SEM. *, P

    Article Snippet: The human K-ras cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using mRNA from SW480 cells that have homozygous mutation at codon 12 of KRAS gene (G12V). pCSGW K-ras V12 or empty vector were introduced with packaging plasmid pCMV-dR8.91 and envelope plasmid pMD2.G into HEK293T cells by transfection with the Fugene6 regent (Roche, Indianapolis, IN).

    Techniques: Quantitative RT-PCR, Transfection, Construct

    Induction of VEGF under hypoxic conditions is suppressed by inhibition of c-Src. A and C , Relative mRNA levels of VEGF, as evaluated by quantitative RT-PCR, in Caco2 and HT29 cells pretreated with 10 µM PP2 ( A ) or transiently transfected with a Src-specific siRNA construct ( C ), and exposed to normoxia or hypoxia for 24 hours. The data are expressed as fold change as compared to control cells in normoxia, normalized to 1. *, P

    Journal: PLoS ONE

    Article Title: Hypoxia Activates the K-Ras Proto-Oncogene to Stimulate Angiogenesis and Inhibit Apoptosis in Colon Cancer Cells

    doi: 10.1371/journal.pone.0010966

    Figure Lengend Snippet: Induction of VEGF under hypoxic conditions is suppressed by inhibition of c-Src. A and C , Relative mRNA levels of VEGF, as evaluated by quantitative RT-PCR, in Caco2 and HT29 cells pretreated with 10 µM PP2 ( A ) or transiently transfected with a Src-specific siRNA construct ( C ), and exposed to normoxia or hypoxia for 24 hours. The data are expressed as fold change as compared to control cells in normoxia, normalized to 1. *, P

    Article Snippet: The human K-ras cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using mRNA from SW480 cells that have homozygous mutation at codon 12 of KRAS gene (G12V). pCSGW K-ras V12 or empty vector were introduced with packaging plasmid pCMV-dR8.91 and envelope plasmid pMD2.G into HEK293T cells by transfection with the Fugene6 regent (Roche, Indianapolis, IN).

    Techniques: Inhibition, Quantitative RT-PCR, Transfection, Construct

    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in Igκ wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative PCR via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.

    Journal: Frontiers in Immunology

    Article Title: A Novel Pax5-Binding Regulatory Element in the Ig? Locus

    doi: 10.3389/fimmu.2014.00240

    Figure Lengend Snippet: Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in Igκ wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative PCR via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.

    Article Snippet: WT and ΔDm rearranged Igκ alleles were amplified with Vκ-Degenerate 5′-GTCCCTGCCAGGTTYAGTGGCAGTGGRTCWRGGAC-3′ and R3-1 5′-CAGACCCTGGTCTAATGGTTTGTAACCACATGGG-3′ primers using high fidelity PCR kit (Roche) with an initial denaturation of 4 min at 94°C, followed by 35 cycles of denaturation at 94°C for 15 s and annealing combined with elongation at 68°C for 2 min. 3′ A-overhang nucleotides were added by 20 min incubation with Taq polymerase and ATP at 72°C.

    Techniques: Binding Assay, Sequencing, Electro Mobility Shift Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Footprinting, Labeling, Chromatin Immunoprecipitation, Cell Culture, Real-time Polymerase Chain Reaction, Immunoprecipitation, Polymerase Chain Reaction

    Heat shock and cadmium sulfate change the alternative splicing pattern of the adenovirus E1A minigene. (A) Schematic representation of the alternative spliced isoforms of E1A pre-mRNA. The major isoforms 9S, 12S, and 13S are generated by alternative selection of the 5′ splice sites. The minor isoforms 10S and 11S involve the usage of additional internal 3′ splice sites. Sizes, in base pairs, of the corresponding RT-PCR products obtained with the use of the E1A-569 and E1A-1315 primers are shown. (B) In vivo alternative splicing of E1A pre-mRNA is affected by heat shock and by cadmium sulfate. The pCMVE1A plasmid containing the E1A minigene was transfected in HeLa cells. Twenty-four hours after transfection total RNAs were extracted from unstressed cells (37°C) or from cells heat-shocked for 1 h at 42°C. RNAs were also prepared from cells allowed to recover 1, 2, 4, or 6 h at 37°C after heat shock. RNAs were also extracted from cells treated for 3 or 5 h with 30 μM of cadmium sulfate, another induced of HAP bodies. RNA were analyzed by RT-PCR with E1A-569 and E1A-1315 primers. RT-PCR products were resolved on 5% polyacrylamide gel and detected by autoradiography. (C) ImageQuant PhosphorImager quantification of the major E1A isoforms: white bars, light gray bars, and dark gray bars, represent 13S, 12S, and 9S molecules, respectively. Percentages of the three isoforms are the average of at least three independent experiments. Error bars are shown.

    Journal: Molecular Biology of the Cell

    Article Title: Stress-induced Nuclear Bodies Are Sites of Accumulation of Pre-mRNA Processing Factors

    doi:

    Figure Lengend Snippet: Heat shock and cadmium sulfate change the alternative splicing pattern of the adenovirus E1A minigene. (A) Schematic representation of the alternative spliced isoforms of E1A pre-mRNA. The major isoforms 9S, 12S, and 13S are generated by alternative selection of the 5′ splice sites. The minor isoforms 10S and 11S involve the usage of additional internal 3′ splice sites. Sizes, in base pairs, of the corresponding RT-PCR products obtained with the use of the E1A-569 and E1A-1315 primers are shown. (B) In vivo alternative splicing of E1A pre-mRNA is affected by heat shock and by cadmium sulfate. The pCMVE1A plasmid containing the E1A minigene was transfected in HeLa cells. Twenty-four hours after transfection total RNAs were extracted from unstressed cells (37°C) or from cells heat-shocked for 1 h at 42°C. RNAs were also prepared from cells allowed to recover 1, 2, 4, or 6 h at 37°C after heat shock. RNAs were also extracted from cells treated for 3 or 5 h with 30 μM of cadmium sulfate, another induced of HAP bodies. RNA were analyzed by RT-PCR with E1A-569 and E1A-1315 primers. RT-PCR products were resolved on 5% polyacrylamide gel and detected by autoradiography. (C) ImageQuant PhosphorImager quantification of the major E1A isoforms: white bars, light gray bars, and dark gray bars, represent 13S, 12S, and 9S molecules, respectively. Percentages of the three isoforms are the average of at least three independent experiments. Error bars are shown.

    Article Snippet: Portions of the open reading frame of HAP were polymerase chain reaction (PCR)-amplified with Pwo polymerase (Roche Molecular Biochemicals) with the use of the HAP cDNA ( ) as template and suitable primers synthesized on the basis of the cDNA sequence (accession number NM-002967).

    Techniques: Generated, Selection, Reverse Transcription Polymerase Chain Reaction, In Vivo, Plasmid Preparation, Transfection, Autoradiography

    Frequency and relative expression of KIR genes in the rhesus macaque cohort . A) Y-axis indicates the percentage of animals within the cohort (n = 69) that express the indicated KIR gene. Genes not listed were not present in any animal within our cohort. Mamu-KIR2DL04 was excluded since it is not amplified by our pyrosequencing amplicon. B) Graph illustrates the percent of total pyrosequencing reads per animal (n = 61) for each KIR gene. Averages and SEM are represented by error bars. Each animal included had at least 100 sequencing reads. Genotyping results representing less than 1% of total reads in an animal were excluded to mitigate the influence of PCR artifacts. Ambiguous reads matching more than one KIR gene and splice variants were also excluded.

    Journal: BMC Genomics

    Article Title: Characterization of killer immunoglobulin-like receptor genetics and comprehensive genotyping by pyrosequencing in rhesus macaques

    doi: 10.1186/1471-2164-12-295

    Figure Lengend Snippet: Frequency and relative expression of KIR genes in the rhesus macaque cohort . A) Y-axis indicates the percentage of animals within the cohort (n = 69) that express the indicated KIR gene. Genes not listed were not present in any animal within our cohort. Mamu-KIR2DL04 was excluded since it is not amplified by our pyrosequencing amplicon. B) Graph illustrates the percent of total pyrosequencing reads per animal (n = 61) for each KIR gene. Averages and SEM are represented by error bars. Each animal included had at least 100 sequencing reads. Genotyping results representing less than 1% of total reads in an animal were excluded to mitigate the influence of PCR artifacts. Ambiguous reads matching more than one KIR gene and splice variants were also excluded.

    Article Snippet: Emulsion PCR, Roche/454 Titanium amplicon pyrosequencing, image processing, and base calling were performed according to the manufacturer's instructions (Roche/454 Life Sciences, Branford, CT) at the University of Illinois at Urbana-Champaign High-Throughput Sequencing Center.

    Techniques: Expressing, Amplification, Sequencing, Polymerase Chain Reaction

    KIR 454 Titanium Amplicon Primer Design . A) Schematic representation of macaque KIR3D and KIR2D molecules showing domain structure. B) Variability plot for a cDNA sequence alignment of all published KIR3DL alleles. PCR primer sites are indicated along with the region amplified by PCR. Note that 454 sequencing reads span the D1, D2, and stem (ST) regions. The signal sequence (SS), D0, transmembrane region (T), and cytoplasmic region (C) are not amplified by our PCR primers.

    Journal: BMC Genomics

    Article Title: Characterization of killer immunoglobulin-like receptor genetics and comprehensive genotyping by pyrosequencing in rhesus macaques

    doi: 10.1186/1471-2164-12-295

    Figure Lengend Snippet: KIR 454 Titanium Amplicon Primer Design . A) Schematic representation of macaque KIR3D and KIR2D molecules showing domain structure. B) Variability plot for a cDNA sequence alignment of all published KIR3DL alleles. PCR primer sites are indicated along with the region amplified by PCR. Note that 454 sequencing reads span the D1, D2, and stem (ST) regions. The signal sequence (SS), D0, transmembrane region (T), and cytoplasmic region (C) are not amplified by our PCR primers.

    Article Snippet: Emulsion PCR, Roche/454 Titanium amplicon pyrosequencing, image processing, and base calling were performed according to the manufacturer's instructions (Roche/454 Life Sciences, Branford, CT) at the University of Illinois at Urbana-Champaign High-Throughput Sequencing Center.

    Techniques: Amplification, Sequencing, Polymerase Chain Reaction