The Experts below are selected from a list of 26511 Experts worldwide ranked by ideXlab platform

P M Williams - One of the best experts on this subject based on the ideXlab platform.

  • development and validation of real time quantitative reverse transcriptase polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro
    Analytical Biochemistry, 1999
    Co-Authors: J Winer, C K S Jung, I Shackel, P M Williams
    Abstract:

    In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA Contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.

  • development and validation of real time quantitative reverse transcriptase polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro
    Analytical Biochemistry, 1999
    Co-Authors: J Winer, C K S Jung, I Shackel, P M Williams
    Abstract:

    In this article we present validation of a real-time RT–PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in anin vitromodel of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT–PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA Contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the ΔΔCtmethod. Real-time quantitative RT–PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.

Peter Lindblad - One of the best experts on this subject based on the ideXlab platform.

  • analysis of current and alternative phenol based rna extraction methodologies for cyanobacteria
    BMC Molecular Biology, 2009
    Co-Authors: Fernando Lopes Pinto, Anders Thapper, Wolfgang Sontheim, Peter Lindblad
    Abstract:

    Background The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA Contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.

  • analysis of current and alternative phenol based rna extraction methodologies for cyanobacteria
    BMC Molecular Biology, 2009
    Co-Authors: Fernando Lopes Pinto, Anders Thapper, Wolfgang Sontheim, Peter Lindblad
    Abstract:

    The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA Contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction. With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution. It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.

J Winer - One of the best experts on this subject based on the ideXlab platform.

  • development and validation of real time quantitative reverse transcriptase polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro
    Analytical Biochemistry, 1999
    Co-Authors: J Winer, C K S Jung, I Shackel, P M Williams
    Abstract:

    In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA Contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.

  • development and validation of real time quantitative reverse transcriptase polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro
    Analytical Biochemistry, 1999
    Co-Authors: J Winer, C K S Jung, I Shackel, P M Williams
    Abstract:

    In this article we present validation of a real-time RT–PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in anin vitromodel of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT–PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA Contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the ΔΔCtmethod. Real-time quantitative RT–PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.

Fernando Lopes Pinto - One of the best experts on this subject based on the ideXlab platform.

  • analysis of current and alternative phenol based rna extraction methodologies for cyanobacteria
    BMC Molecular Biology, 2009
    Co-Authors: Fernando Lopes Pinto, Anders Thapper, Wolfgang Sontheim, Peter Lindblad
    Abstract:

    Background The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA Contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.

  • analysis of current and alternative phenol based rna extraction methodologies for cyanobacteria
    BMC Molecular Biology, 2009
    Co-Authors: Fernando Lopes Pinto, Anders Thapper, Wolfgang Sontheim, Peter Lindblad
    Abstract:

    The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA Contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction. With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution. It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.

C K S Jung - One of the best experts on this subject based on the ideXlab platform.

  • development and validation of real time quantitative reverse transcriptase polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro
    Analytical Biochemistry, 1999
    Co-Authors: J Winer, C K S Jung, I Shackel, P M Williams
    Abstract:

    In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA Contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.

  • development and validation of real time quantitative reverse transcriptase polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro
    Analytical Biochemistry, 1999
    Co-Authors: J Winer, C K S Jung, I Shackel, P M Williams
    Abstract:

    In this article we present validation of a real-time RT–PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in anin vitromodel of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT–PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA Contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the ΔΔCtmethod. Real-time quantitative RT–PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.