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

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Analytical Biochemistry (Impact Factor: 2.22). 06/1999; 270(1):41-9. DOI: 10.1006/abio.1999.4085
Source: PubMed


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.

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    • "A thermal denaturing step to generate the dissociation curves was included to verify amplification specificity. All reactions were run in triplicate, and the results were analyzed by the 2 -ΔΔCT method [61, 62]. Student's t-test in EXCEL was used to analyze the qRT- PCR data of miRNA comparisons between nonviruliferous and viruliferous whiteflies. "

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    • "In the current study, porcine GLP2R cDNA was cloned using the rapid amplification cDNA ends (RACE) technique (Yeku and Frohman, 2011). Bioinformatics and phylogenetic tree analyses were performed based on porcine GLP2R amino acid sequence, and real-time quantitative PCR (Winer et al., 1999; Jin et al., 2013) was adopted to inspect GLP2R expression patterns in ten tissue types from two pig breeds. The purpose of the study is to lay a foundation for the study of GLP2R gene in the formation of IMF in the porcine. "
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    Preview · Article · Oct 2015 · Genetics and molecular research: GMR
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    • "The Tth111I polymorphism of the NR3C1 gene was genotyped using the PCR-HRM method with application of LightScanner® 32 System [19] [20] [21] [22] [40] [52] [53]. Expression of TGFβ1 gene was measured with real-time quantitative PCR (qRT-PCR) method and the 2−ΔΔCT method (ddCT — delta delta cycle threshold values) [54] [55] [56] [57]. Genotyping and qRT-PCR were performed by two investigators who were unaware of the participants' phenotypes. "
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