Development and Validation of Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction for Monitoring Gene Expression in Cardiac Myocytesin Vitro
Genentech Inc., One DNA Way, South San Francisco, California 94080, USA. Analytical Biochemistry
(Impact Factor: 2.22).
06/1999; 270(1):41-9. DOI: 10.1006/abio.1999.4085
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.
Available from: virologyj.biomedcentral.com
- "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. "
Available from: funpecrp.com.br
- "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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ABSTRACT: Glucagon-like peptide-2 receptor (GLP2R), a member of the G-protein-coupled receptor family, plays an important role in intramuscular fat formation. Little is known, however, about porcine GLP2R. In the present study, GLP2R was cloned, and its expression in pig muscle characterized. By rapid amplification of cDNA ends, gene sequence was obtained from Shaziling pigs. Full-length cDNA was 1868 bp, including an open reading frame 1665 bp in length, encoding 554 amino acids, and 203 bp at the 3' end. The GLP2R homology between porcine and other species was performed using bioinformatics techniques to construct a phylogenetic tree. Porcine GLP2R was most closely related to those from Orcinus orca and Ovis aries，and most distantly related to those from Chrysemys picta, Taeniopygia guttata, and Falco peregrinus. Real-time PCR analysis showed expression of porcine GLP2R in 10 different tissues from 25-day-old Yorkshire and Shaziling piglets, with expression levels being highest in the longissimus dorsi muscle and lowest in kidney. For each pig breed, expression level in longissimus dorsi muscle was highest among ten tissues (P < 0.05). Between the two breeds, GLP2R expression levels were significant in pancreas, the crureus and longissimus dorsi muscles (P < 0.05). A single SNP of porcine GLP2R, A343G, was identified, and genotypes were determined by PCR-RFLP. This study provides an insight into the function of GLP2R in swine.
Available from: Piotr Kuna
- "The Tth111I polymorphism of the NR3C1 gene was genotyped using the PCR-HRM method with application of LightScanner® 32 System       . 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)    . Genotyping and qRT-PCR were performed by two investigators who were unaware of the participants' phenotypes. "
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ABSTRACT: Personal and environmental factors might have an impact on strategies of coping with stress and temperamental traits according to the Regulative Theory of Temperament in asthmatic patients. They can modify the clinical picture, the course of a disease and effectiveness of treatment. Personal variables are key factors in determining formal characteristic of behavior and effective management method in asthmatic patients.
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