input dna input  (Roche)


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    Structured Review

    Roche input dna input
    Input Dna Input, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/input dna input/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    input dna input - by Bioz Stars, 2021-06
    86/100 stars

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    Labeling:

    Article Title: Polyploid cells rewire DNA damage response networks to overcome replication stress-induced barriers for tumour progression
    Article Snippet: Enriched methylated and input genomic DNA samples were quantitatively amplified by real-time PCR and labeled with Cy3 and Cy5, respectively. .. The labeled DNA fragments were hybridized to the Nimblegen HG18 CpG Promoter array (Roche Nimblegen). .. Normalized hybridization signals were analyzed with SignalMap (Nimblegen).

    Sequencing:

    Article Title: Circulating tumor DNA profile recognizes transformation to castration-resistant neuroendocrine prostate cancer
    Article Snippet: Lysates were then transferred to the first well of a multiwall cartridge prefilled with reagents, 65 μL elution buffer was loaded, and the automated system successfully isolated high-concentration genomic DNA. cfDNA was quantified with Qubit and quality was also assessed using Agilent’s High Sensitivity DNA kit. .. Whole-exome sequencing of ctDNA and matched PBMC DNABased on the expected circulating DNA fragment size distribution and the range of input DNA identified from each plasma sample, we opted for the Roche SeqCap EZ Library SR for library preparation. .. Germline genomic DNA was sheared using the Covaris E220 Evolution instrument.

    Article Title: Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts
    Article Snippet: DNA was extracted using the Qiagen PCR-clean up columns. .. The immunoprecipitated DNAs (output) were quantitated by real time PCR (LightCycler 480 from Roche with the products and instructions recommended by the supplier) using three sets of primers located along the PMA1 or PGK1 coding sequence (described in Supplementary Table S5 ) and normalized to a 1/200 dilution of input DNA. .. Bacterial Rho factor induces the production of aberrant transcripts in yeast In a recent study , we reported that the conditional expression of the bacterial Rho factor in the yeast nucleus leads to a dose-dependent growth defect phenotype that stems from its interference in mRNP biogenesis.

    Purification:

    Article Title: Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology
    Article Snippet: Bound fractions, and an amount corresponding to 5% of previously saved inputs, were de-crosslinked, purified, and quantified (triplicate readings) as described above. .. Locus-specific analysis of immunoselected (bound) DNA Purified DNA from bound and 5% input fractions was analyzed in triplicate by real-time quantitative PCR (qPCR) using the Fast Start SYBR Green Master Mix (Roche, Mannheim, Germany) and the Rotor-Gene 6000 robocycler (Corbett Life Science, Sydney, Australia) as already reported [ ]. .. Purified DNA from bound and 5% input fractions was analyzed in triplicate by real-time quantitative PCR (qPCR) using the Fast Start SYBR Green Master Mix (Roche, Mannheim, Germany) and the Rotor-Gene 6000 robocycler (Corbett Life Science, Sydney, Australia) as already reported [ ].

    Real-time Polymerase Chain Reaction:

    Article Title: Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology
    Article Snippet: Bound fractions, and an amount corresponding to 5% of previously saved inputs, were de-crosslinked, purified, and quantified (triplicate readings) as described above. .. Locus-specific analysis of immunoselected (bound) DNA Purified DNA from bound and 5% input fractions was analyzed in triplicate by real-time quantitative PCR (qPCR) using the Fast Start SYBR Green Master Mix (Roche, Mannheim, Germany) and the Rotor-Gene 6000 robocycler (Corbett Life Science, Sydney, Australia) as already reported [ ]. .. Purified DNA from bound and 5% input fractions was analyzed in triplicate by real-time quantitative PCR (qPCR) using the Fast Start SYBR Green Master Mix (Roche, Mannheim, Germany) and the Rotor-Gene 6000 robocycler (Corbett Life Science, Sydney, Australia) as already reported [ ].

    Article Title: Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts
    Article Snippet: DNA was extracted using the Qiagen PCR-clean up columns. .. The immunoprecipitated DNAs (output) were quantitated by real time PCR (LightCycler 480 from Roche with the products and instructions recommended by the supplier) using three sets of primers located along the PMA1 or PGK1 coding sequence (described in Supplementary Table S5 ) and normalized to a 1/200 dilution of input DNA. .. Bacterial Rho factor induces the production of aberrant transcripts in yeast In a recent study , we reported that the conditional expression of the bacterial Rho factor in the yeast nucleus leads to a dose-dependent growth defect phenotype that stems from its interference in mRNP biogenesis.

    Article Title: The conformational flexibility of the C-terminus of histone H4 promotes histone octamer and nucleosome stability and yeast viability
    Article Snippet: The DNA was purified using the MiniElute DNA Purification kit from Qiagen (catalog no. 28004). .. ChIP quantification was performed via real-time PCR with a Roche Applied Sciences LightCycler 480 and the LightCycler 480 SYBR Green I Master Mix kit (Roche catalog no. 04707516001). .. Standard curves were generated using Input DNA dilutions of 1:10, 1:100, 1:1,000 and 1:10,000.

    SYBR Green Assay:

    Article Title: Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology
    Article Snippet: Bound fractions, and an amount corresponding to 5% of previously saved inputs, were de-crosslinked, purified, and quantified (triplicate readings) as described above. .. Locus-specific analysis of immunoselected (bound) DNA Purified DNA from bound and 5% input fractions was analyzed in triplicate by real-time quantitative PCR (qPCR) using the Fast Start SYBR Green Master Mix (Roche, Mannheim, Germany) and the Rotor-Gene 6000 robocycler (Corbett Life Science, Sydney, Australia) as already reported [ ]. .. Purified DNA from bound and 5% input fractions was analyzed in triplicate by real-time quantitative PCR (qPCR) using the Fast Start SYBR Green Master Mix (Roche, Mannheim, Germany) and the Rotor-Gene 6000 robocycler (Corbett Life Science, Sydney, Australia) as already reported [ ].

    Article Title: Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1
    Article Snippet: The ChIP DNA and input DNA obtained were then used for qPCR analyses with the ‘DNA Standards, design and analysis template’ provided by ChIP-IT qPCR Analysis kit. .. The qPCRs were performed using a Roche LightCycler 480 and LightCycler 480 SYBR Green I Master Kit Mix (Roche Diagnostics), with primers located in the hPCFT core promoter region that includes putative binding sites for NRF-1, KLF15 or Sp1. ..

    Article Title: The conformational flexibility of the C-terminus of histone H4 promotes histone octamer and nucleosome stability and yeast viability
    Article Snippet: The DNA was purified using the MiniElute DNA Purification kit from Qiagen (catalog no. 28004). .. ChIP quantification was performed via real-time PCR with a Roche Applied Sciences LightCycler 480 and the LightCycler 480 SYBR Green I Master Mix kit (Roche catalog no. 04707516001). .. Standard curves were generated using Input DNA dilutions of 1:10, 1:100, 1:1,000 and 1:10,000.

    Polymerase Chain Reaction:

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare
    Article Snippet: We also obtained genomic DNA samples from cerebellum autopsy tissues from three FRDA patients (tissues were registered with the HTA under Brunel University Licensing number 12543) and five ear biopsies from previously described GAA repeat expansion-based Y47R and YG8sR FRDA mouse models ( ) (animal procedures were carried out in accordance with the UK Home Office “Animals (Scientific Procedures) Act 1986” and with approval from the Brunel University London Animal Welfare and Ethical Review Board). .. We then performed long-range PCR of the samples (approximately 100 ng input DNA) using either the Expand High Fidelity PCR System, dNTPack (Roche), or the Long Range PCR Kit (Qiagen) together with GAA-B-F (5′-AATGGATTTCCTGGCAGGACGC-3′) and GAA-B-R (5′-GCATTGGGCGATCTTGGCTTAA-3′) primers as previously described ( ). .. The thermocycling conditions used were (i) Roche Kit: 94°C for 2 min; 10 cycles of 94°C for 10 s, 60°C for 30 s, 68°C for 45 s; 20 cycles of 94°C for 10 s, 60°C for 30 s, 68°C for 1 min with 20 s increments; and a final cycle of 68°C for 10 mins, or (ii) Qiagen Kit: 93°C for 3 min; 35 cycles of 93°C for 15 s, 62°C for 30 s, 68°C for 5 min, and a final cycle of 68°C for 10 min.

    Immunoprecipitation:

    Article Title: Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts
    Article Snippet: DNA was extracted using the Qiagen PCR-clean up columns. .. The immunoprecipitated DNAs (output) were quantitated by real time PCR (LightCycler 480 from Roche with the products and instructions recommended by the supplier) using three sets of primers located along the PMA1 or PGK1 coding sequence (described in Supplementary Table S5 ) and normalized to a 1/200 dilution of input DNA. .. Bacterial Rho factor induces the production of aberrant transcripts in yeast In a recent study , we reported that the conditional expression of the bacterial Rho factor in the yeast nucleus leads to a dose-dependent growth defect phenotype that stems from its interference in mRNP biogenesis.

    Binding Assay:

    Article Title: Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1
    Article Snippet: The ChIP DNA and input DNA obtained were then used for qPCR analyses with the ‘DNA Standards, design and analysis template’ provided by ChIP-IT qPCR Analysis kit. .. The qPCRs were performed using a Roche LightCycler 480 and LightCycler 480 SYBR Green I Master Kit Mix (Roche Diagnostics), with primers located in the hPCFT core promoter region that includes putative binding sites for NRF-1, KLF15 or Sp1. ..

    Chromatin Immunoprecipitation:

    Article Title: The conformational flexibility of the C-terminus of histone H4 promotes histone octamer and nucleosome stability and yeast viability
    Article Snippet: The DNA was purified using the MiniElute DNA Purification kit from Qiagen (catalog no. 28004). .. ChIP quantification was performed via real-time PCR with a Roche Applied Sciences LightCycler 480 and the LightCycler 480 SYBR Green I Master Mix kit (Roche catalog no. 04707516001). .. Standard curves were generated using Input DNA dilutions of 1:10, 1:100, 1:1,000 and 1:10,000.

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    Roche dna input
    Optimization of virial transduction conditions for human MDA-MB-231 cells ( A ) Schematic representation of the non-invasive apoptosis detection sensor (NIADS). The fusion proteins of pepA-Nluc and pepB-Cluc fragment were linked with 3Xcaspase-3 cleavage sequences (DEVD). Once the cell is undergoing the apoptosis event, activated caspase-3 recognizes the DEVD sequence and cuts the fusion protein into two fragments. The proteins of pepA and pepB are known to have strong association force and therefore enable reconstitution of full-length luciferase. The bioluminescence activity can be further detected with substrate (Luciferin) addition; ( B ) NIADS <t>DNA</t> sequence was cloned into lentivirus plasmid and generated virus particles in medium. The virus titration from 10–40 μL of both RFP (red fluorescent protein) or NIADS was added in the culture medium of luciferase stable expressing MDA-MB-231 cells for three days. The cells were harvested and immunoblotted to luciferase antibody, whereas the full length of firefly luciferase was determined as protein loading control; ( C ) The virus titration from 10 to 40 μL RFP virus-containing medium was measured on MDA-MB-231 cells for three days. The image of RFP expression was captured under florescence microscopy. Scale bar: 50 μM; ( D ) Absolute <t>qPCR</t> analysis was measured by serial copy number of GUS (β-glucuronidase)-containing TA vector from a range of 10 2 (purple line) to 108 (blue line)/μL as a standard curve. The gene amplification curves were also determined for standard error and gene amplification efficiency. The qPCR efficiency analysis for standard curve is calculated by the formula of Efficiency = 10 −1/slope , whereas qPCR unexplained variance of R 2 value was calculated by the formula of R 2 = 1 − Error; ( E ) The purified and concentrated RFP and NIADS lentivirus were determined as virus copy number/μL by absolute qPCR analysis. Data is presented as the mean and standard error; ( F ) The purified and concentrated RFP lentivirus were measured as virus copy number by qPCR analysis. MDA-MB-231 cells were seeded in a 6-cm dish and infected with 6-, 12-, 30-, 60-, 120-, 240-fold concentrations of virus to MDA-MB-231 cell number for three days. The RFP-positive (infected) cell population was assessed using flow cytometry; ( G ) Linear curve comparison of virus input and the RFP-positive cell population; ( H ) The MOI (240-fold as MOI = 1, multiplicity of infection) from 0.1 to 6 of NIADS contain lentivirus were used on MDA-MB-231 cell. The NIADS fusion protein was immunoblotted by luciferase antibody.
    Dna Input, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna input/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna input - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

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    Roche cobas ampliprep instrument accepts input
    (A) Minimum control requirements with respect to batch size for the Abbott m 2000 RealTi m e and Roche <t>COBAS</t> <t>AmpliPrep/COBAS</t> TaqMan systems. The Abbott m 2000 RealTi m e system can process 96 samples in one run, while the Roche COBAS AmpliPrep/COBAS TaqMan
    Cobas Ampliprep Instrument Accepts Input, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cobas ampliprep instrument accepts input/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cobas ampliprep instrument accepts input - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    86
    Roche input dna
    Identification of methylation sensitive <t>miRNA</t> ( a ) Flowchart representing study design. ( b ) Integrated heatmap and two-dimensional hierarchical clustering for 67 methylation sensitive miRNAs. Tumors are clustered horizontally while miRNA are clustered vertically. Characteristics of the tumors are displayed at the top of the heat map. The heat map is based on both levels of miRNA expression and levels of <t>DNA</t> methylation at each locus, with a key to the color coding presented in the upper left corner. The colored vertical bar on the left designates miRNAs from the same cluster which display similarity in expression levels due to being under the control of a common upstream site of methylation. The table on the right designates miRNAs that are associated with poor OS/EFS when under-expressed in tumours (black, n = 28) or over-expressed in tumors (grey, n = 10). miRNAs that are up-regulated in response to the demethylating agent, 5-aza-cytidine, in four different cell lines are designated in the table. ( c ) The distribution of all methylation peaks within 10kb of CpG Islands. The majority of peaks occur within regions defined as CpG shores.
    Input Dna, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/input dna/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    input dna - by Bioz Stars, 2021-06
    86/100 stars
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    Optimization of virial transduction conditions for human MDA-MB-231 cells ( A ) Schematic representation of the non-invasive apoptosis detection sensor (NIADS). The fusion proteins of pepA-Nluc and pepB-Cluc fragment were linked with 3Xcaspase-3 cleavage sequences (DEVD). Once the cell is undergoing the apoptosis event, activated caspase-3 recognizes the DEVD sequence and cuts the fusion protein into two fragments. The proteins of pepA and pepB are known to have strong association force and therefore enable reconstitution of full-length luciferase. The bioluminescence activity can be further detected with substrate (Luciferin) addition; ( B ) NIADS DNA sequence was cloned into lentivirus plasmid and generated virus particles in medium. The virus titration from 10–40 μL of both RFP (red fluorescent protein) or NIADS was added in the culture medium of luciferase stable expressing MDA-MB-231 cells for three days. The cells were harvested and immunoblotted to luciferase antibody, whereas the full length of firefly luciferase was determined as protein loading control; ( C ) The virus titration from 10 to 40 μL RFP virus-containing medium was measured on MDA-MB-231 cells for three days. The image of RFP expression was captured under florescence microscopy. Scale bar: 50 μM; ( D ) Absolute qPCR analysis was measured by serial copy number of GUS (β-glucuronidase)-containing TA vector from a range of 10 2 (purple line) to 108 (blue line)/μL as a standard curve. The gene amplification curves were also determined for standard error and gene amplification efficiency. The qPCR efficiency analysis for standard curve is calculated by the formula of Efficiency = 10 −1/slope , whereas qPCR unexplained variance of R 2 value was calculated by the formula of R 2 = 1 − Error; ( E ) The purified and concentrated RFP and NIADS lentivirus were determined as virus copy number/μL by absolute qPCR analysis. Data is presented as the mean and standard error; ( F ) The purified and concentrated RFP lentivirus were measured as virus copy number by qPCR analysis. MDA-MB-231 cells were seeded in a 6-cm dish and infected with 6-, 12-, 30-, 60-, 120-, 240-fold concentrations of virus to MDA-MB-231 cell number for three days. The RFP-positive (infected) cell population was assessed using flow cytometry; ( G ) Linear curve comparison of virus input and the RFP-positive cell population; ( H ) The MOI (240-fold as MOI = 1, multiplicity of infection) from 0.1 to 6 of NIADS contain lentivirus were used on MDA-MB-231 cell. The NIADS fusion protein was immunoblotted by luciferase antibody.

    Journal: International Journal of Molecular Sciences

    Article Title: The Application of Non-Invasive Apoptosis Detection Sensor (NIADS) on Histone Deacetylation Inhibitor (HDACi)-Induced Breast Cancer Cell Death

    doi: 10.3390/ijms19020452

    Figure Lengend Snippet: Optimization of virial transduction conditions for human MDA-MB-231 cells ( A ) Schematic representation of the non-invasive apoptosis detection sensor (NIADS). The fusion proteins of pepA-Nluc and pepB-Cluc fragment were linked with 3Xcaspase-3 cleavage sequences (DEVD). Once the cell is undergoing the apoptosis event, activated caspase-3 recognizes the DEVD sequence and cuts the fusion protein into two fragments. The proteins of pepA and pepB are known to have strong association force and therefore enable reconstitution of full-length luciferase. The bioluminescence activity can be further detected with substrate (Luciferin) addition; ( B ) NIADS DNA sequence was cloned into lentivirus plasmid and generated virus particles in medium. The virus titration from 10–40 μL of both RFP (red fluorescent protein) or NIADS was added in the culture medium of luciferase stable expressing MDA-MB-231 cells for three days. The cells were harvested and immunoblotted to luciferase antibody, whereas the full length of firefly luciferase was determined as protein loading control; ( C ) The virus titration from 10 to 40 μL RFP virus-containing medium was measured on MDA-MB-231 cells for three days. The image of RFP expression was captured under florescence microscopy. Scale bar: 50 μM; ( D ) Absolute qPCR analysis was measured by serial copy number of GUS (β-glucuronidase)-containing TA vector from a range of 10 2 (purple line) to 108 (blue line)/μL as a standard curve. The gene amplification curves were also determined for standard error and gene amplification efficiency. The qPCR efficiency analysis for standard curve is calculated by the formula of Efficiency = 10 −1/slope , whereas qPCR unexplained variance of R 2 value was calculated by the formula of R 2 = 1 − Error; ( E ) The purified and concentrated RFP and NIADS lentivirus were determined as virus copy number/μL by absolute qPCR analysis. Data is presented as the mean and standard error; ( F ) The purified and concentrated RFP lentivirus were measured as virus copy number by qPCR analysis. MDA-MB-231 cells were seeded in a 6-cm dish and infected with 6-, 12-, 30-, 60-, 120-, 240-fold concentrations of virus to MDA-MB-231 cell number for three days. The RFP-positive (infected) cell population was assessed using flow cytometry; ( G ) Linear curve comparison of virus input and the RFP-positive cell population; ( H ) The MOI (240-fold as MOI = 1, multiplicity of infection) from 0.1 to 6 of NIADS contain lentivirus were used on MDA-MB-231 cell. The NIADS fusion protein was immunoblotted by luciferase antibody.

    Article Snippet: Luciferase gene expression from the qPCR analysis was normalized to GUS (β-glucuronidase) expression as an indicator of DNA input using the built-in Roche LightCycler Software, version 4 (Roche Molecular Biochemicals, Mannheim, German).

    Techniques: Transduction, Multiple Displacement Amplification, Sequencing, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation, Generated, Titration, Expressing, Microscopy, Real-time Polymerase Chain Reaction, Amplification, Purification, Infection, Flow Cytometry, Cytometry

    Optimization of virial transduction conditions for human MDA-MB-231 cells ( A ) Schematic representation of the non-invasive apoptosis detection sensor (NIADS). The fusion proteins of pepA-Nluc and pepB-Cluc fragment were linked with 3Xcaspase-3 cleavage sequences (DEVD). Once the cell is undergoing the apoptosis event, activated caspase-3 recognizes the DEVD sequence and cuts the fusion protein into two fragments. The proteins of pepA and pepB are known to have strong association force and therefore enable reconstitution of full-length luciferase. The bioluminescence activity can be further detected with substrate (Luciferin) addition; ( B ) NIADS DNA sequence was cloned into lentivirus plasmid and generated virus particles in medium. The virus titration from 10–40 μL of both RFP (red fluorescent protein) or NIADS was added in the culture medium of luciferase stable expressing MDA-MB-231 cells for three days. The cells were harvested and immunoblotted to luciferase antibody, whereas the full length of firefly luciferase was determined as protein loading control; ( C ) The virus titration from 10 to 40 μL RFP virus-containing medium was measured on MDA-MB-231 cells for three days. The image of RFP expression was captured under florescence microscopy. Scale bar: 50 μM; ( D ) Absolute qPCR analysis was measured by serial copy number of GUS (β-glucuronidase)-containing TA vector from a range of 10 2 (purple line) to 108 (blue line)/μL as a standard curve. The gene amplification curves were also determined for standard error and gene amplification efficiency. The qPCR efficiency analysis for standard curve is calculated by the formula of Efficiency = 10 −1/slope , whereas qPCR unexplained variance of R 2 value was calculated by the formula of R 2 = 1 − Error; ( E ) The purified and concentrated RFP and NIADS lentivirus were determined as virus copy number/μL by absolute qPCR analysis. Data is presented as the mean and standard error; ( F ) The purified and concentrated RFP lentivirus were measured as virus copy number by qPCR analysis. MDA-MB-231 cells were seeded in a 6-cm dish and infected with 6-, 12-, 30-, 60-, 120-, 240-fold concentrations of virus to MDA-MB-231 cell number for three days. The RFP-positive (infected) cell population was assessed using flow cytometry; ( G ) Linear curve comparison of virus input and the RFP-positive cell population; ( H ) The MOI (240-fold as MOI = 1, multiplicity of infection) from 0.1 to 6 of NIADS contain lentivirus were used on MDA-MB-231 cell. The NIADS fusion protein was immunoblotted by luciferase antibody.

    Journal: International Journal of Molecular Sciences

    Article Title: The Application of Non-Invasive Apoptosis Detection Sensor (NIADS) on Histone Deacetylation Inhibitor (HDACi)-Induced Breast Cancer Cell Death

    doi: 10.3390/ijms19020452

    Figure Lengend Snippet: Optimization of virial transduction conditions for human MDA-MB-231 cells ( A ) Schematic representation of the non-invasive apoptosis detection sensor (NIADS). The fusion proteins of pepA-Nluc and pepB-Cluc fragment were linked with 3Xcaspase-3 cleavage sequences (DEVD). Once the cell is undergoing the apoptosis event, activated caspase-3 recognizes the DEVD sequence and cuts the fusion protein into two fragments. The proteins of pepA and pepB are known to have strong association force and therefore enable reconstitution of full-length luciferase. The bioluminescence activity can be further detected with substrate (Luciferin) addition; ( B ) NIADS DNA sequence was cloned into lentivirus plasmid and generated virus particles in medium. The virus titration from 10–40 μL of both RFP (red fluorescent protein) or NIADS was added in the culture medium of luciferase stable expressing MDA-MB-231 cells for three days. The cells were harvested and immunoblotted to luciferase antibody, whereas the full length of firefly luciferase was determined as protein loading control; ( C ) The virus titration from 10 to 40 μL RFP virus-containing medium was measured on MDA-MB-231 cells for three days. The image of RFP expression was captured under florescence microscopy. Scale bar: 50 μM; ( D ) Absolute qPCR analysis was measured by serial copy number of GUS (β-glucuronidase)-containing TA vector from a range of 10 2 (purple line) to 108 (blue line)/μL as a standard curve. The gene amplification curves were also determined for standard error and gene amplification efficiency. The qPCR efficiency analysis for standard curve is calculated by the formula of Efficiency = 10 −1/slope , whereas qPCR unexplained variance of R 2 value was calculated by the formula of R 2 = 1 − Error; ( E ) The purified and concentrated RFP and NIADS lentivirus were determined as virus copy number/μL by absolute qPCR analysis. Data is presented as the mean and standard error; ( F ) The purified and concentrated RFP lentivirus were measured as virus copy number by qPCR analysis. MDA-MB-231 cells were seeded in a 6-cm dish and infected with 6-, 12-, 30-, 60-, 120-, 240-fold concentrations of virus to MDA-MB-231 cell number for three days. The RFP-positive (infected) cell population was assessed using flow cytometry; ( G ) Linear curve comparison of virus input and the RFP-positive cell population; ( H ) The MOI (240-fold as MOI = 1, multiplicity of infection) from 0.1 to 6 of NIADS contain lentivirus were used on MDA-MB-231 cell. The NIADS fusion protein was immunoblotted by luciferase antibody.

    Article Snippet: Luciferase gene expression from the qPCR analysis was normalized to GUS (β-glucuronidase) expression as an indicator of DNA input using the built-in Roche LightCycler Software, version 4 (Roche Molecular Biochemicals, Mannheim, German).

    Techniques: Transduction, Multiple Displacement Amplification, Sequencing, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation, Generated, Titration, Expressing, Microscopy, Real-time Polymerase Chain Reaction, Amplification, Purification, Infection, Flow Cytometry, Cytometry

    (A) Minimum control requirements with respect to batch size for the Abbott m 2000 RealTi m e and Roche COBAS AmpliPrep/COBAS TaqMan systems. The Abbott m 2000 RealTi m e system can process 96 samples in one run, while the Roche COBAS AmpliPrep/COBAS TaqMan

    Journal: Journal of Clinical Microbiology

    Article Title: Impact of the New Abbott mPLUS Feature on Clinical Laboratory Efficiencies of Abbott RealTime Assays for Detection of HIV-1, Hepatitis C Virus, Hepatitis B Virus, Chlamydia trachomatis, and Neisseria gonorrhoeae

    doi: 10.1128/JCM.01672-13

    Figure Lengend Snippet: (A) Minimum control requirements with respect to batch size for the Abbott m 2000 RealTi m e and Roche COBAS AmpliPrep/COBAS TaqMan systems. The Abbott m 2000 RealTi m e system can process 96 samples in one run, while the Roche COBAS AmpliPrep/COBAS TaqMan

    Article Snippet: The Roche COBAS AmpliPrep instrument accepts input samples in sealed tubes (S-tubes), and each sample is manually transferred from the primary laboratory tube to the S-tube as part of the preanalytical sample-handling process.

    Techniques:

    Identification of methylation sensitive miRNA ( a ) Flowchart representing study design. ( b ) Integrated heatmap and two-dimensional hierarchical clustering for 67 methylation sensitive miRNAs. Tumors are clustered horizontally while miRNA are clustered vertically. Characteristics of the tumors are displayed at the top of the heat map. The heat map is based on both levels of miRNA expression and levels of DNA methylation at each locus, with a key to the color coding presented in the upper left corner. The colored vertical bar on the left designates miRNAs from the same cluster which display similarity in expression levels due to being under the control of a common upstream site of methylation. The table on the right designates miRNAs that are associated with poor OS/EFS when under-expressed in tumours (black, n = 28) or over-expressed in tumors (grey, n = 10). miRNAs that are up-regulated in response to the demethylating agent, 5-aza-cytidine, in four different cell lines are designated in the table. ( c ) The distribution of all methylation peaks within 10kb of CpG Islands. The majority of peaks occur within regions defined as CpG shores.

    Journal: Oncogene

    Article Title: Modulation of Neuroblastoma Disease Pathogenesis By An Extensive Network of Epigenetically Regulated MicroRNAs

    doi: 10.1038/onc.2012.311

    Figure Lengend Snippet: Identification of methylation sensitive miRNA ( a ) Flowchart representing study design. ( b ) Integrated heatmap and two-dimensional hierarchical clustering for 67 methylation sensitive miRNAs. Tumors are clustered horizontally while miRNA are clustered vertically. Characteristics of the tumors are displayed at the top of the heat map. The heat map is based on both levels of miRNA expression and levels of DNA methylation at each locus, with a key to the color coding presented in the upper left corner. The colored vertical bar on the left designates miRNAs from the same cluster which display similarity in expression levels due to being under the control of a common upstream site of methylation. The table on the right designates miRNAs that are associated with poor OS/EFS when under-expressed in tumours (black, n = 28) or over-expressed in tumors (grey, n = 10). miRNAs that are up-regulated in response to the demethylating agent, 5-aza-cytidine, in four different cell lines are designated in the table. ( c ) The distribution of all methylation peaks within 10kb of CpG Islands. The majority of peaks occur within regions defined as CpG shores.

    Article Snippet: MeDIP and input DNA were hybridized to custom designed miRNA tilling arrays as per the manufacturer’s instructions (Roche NimbleGen, Madison, WI).

    Techniques: Methylation, Expressing, DNA Methylation Assay

    DNA methylation and expressional alterations of miR-340 ( a ) Relative expression of miR-340 in SK-N-BE and SHSY-5Y at 7 days post-ATRA. ( b,c ) Kaplan Meier survival plots for OS ( b ) and EFS ( c ) in 237 neuroblastoma tumors based on miR-340 expression. ( d ) SignalMap image from MeDIP analysis for the miR-340 upstream region. Only the methylation peak highlighted with a bracket (~6 Kb upstream) exhibited significant de-methylation in SK-N-BE cells 7 days post-ATRA. This peak overlaps the predicted TSS for miR-340. ( e ) Scatter plot of methylation vs. miR-340 expression in tumors showing a significant inverse correlation using Pearson’s correlation coefficient. ( f ) Expression of miR-340 following 5’-Aza-2 treatment of SK-N-BE and SHSY-5Y (*P

    Journal: Oncogene

    Article Title: Modulation of Neuroblastoma Disease Pathogenesis By An Extensive Network of Epigenetically Regulated MicroRNAs

    doi: 10.1038/onc.2012.311

    Figure Lengend Snippet: DNA methylation and expressional alterations of miR-340 ( a ) Relative expression of miR-340 in SK-N-BE and SHSY-5Y at 7 days post-ATRA. ( b,c ) Kaplan Meier survival plots for OS ( b ) and EFS ( c ) in 237 neuroblastoma tumors based on miR-340 expression. ( d ) SignalMap image from MeDIP analysis for the miR-340 upstream region. Only the methylation peak highlighted with a bracket (~6 Kb upstream) exhibited significant de-methylation in SK-N-BE cells 7 days post-ATRA. This peak overlaps the predicted TSS for miR-340. ( e ) Scatter plot of methylation vs. miR-340 expression in tumors showing a significant inverse correlation using Pearson’s correlation coefficient. ( f ) Expression of miR-340 following 5’-Aza-2 treatment of SK-N-BE and SHSY-5Y (*P

    Article Snippet: MeDIP and input DNA were hybridized to custom designed miRNA tilling arrays as per the manufacturer’s instructions (Roche NimbleGen, Madison, WI).

    Techniques: DNA Methylation Assay, Expressing, Methylated DNA Immunoprecipitation, Methylation