Regular Article
Development and Validation of Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction for Monitoring Gene Expression in Cardiac Myocytesin Vitro

https://doi.org/10.1006/abio.1999.4085Get rights and content

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.

References (16)

  • K.C. Wollert et al.

    J. Biol. Chem.

    (1996)
  • Z. Sheng et al.

    J. Biol. Chem.

    (1997)
  • H.E. Shubeita et al.

    J. Biol. Chem.

    (1990)
  • D. Pennica et al.

    J. Biol. Chem.

    (1995)
  • K. Iwaki et al.

    J. Biol. Chem.

    (1990)
  • C.A. Heid et al.

    Genome Res.

    (1996)
  • U.E. Gibson et al.

    Genome Res.

    (1996)
There are more references available in the full text version of this article.

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Shu, Chien

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