Assessing gene expression by real-time polymerase chain reaction

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Show simple item record Dang, Ngoc Tran, Paul Colton, Ben Torry, Ronald J. 2007-05-31T15:22:38Z 2007-05-31T15:22:38Z 2007-05-31T15:22:38Z
dc.description.abstract End-point Polymerase Chain Reaction (PCR) is a highly sensitive technique that can be used to assess gene expression in limited tissue/cell samples. However, accurately quantifying gene expression with this method is tedious and inconsistent if not performed properly. Real-time PCR refers to using fluorescent probes that bind double-stranded DNA to monitor DNA product formation during PCR. The number of cycles required for the amplicon fluorescence to reach a threshold level is inversely correlated with the initial amount of target sequence present. The present study compared end-point and real-time PCR techniques to determine whether hypoxia alters growth factor mRNA expression in cardiomyocytes. We chose to assess placenta growth factor (PIGF) expression, an important angiogenic factor with normally low level expression outside of the placenta. End-point RT-PCR showed that cardiomyocytes express PIGF and its receptor, VEGFR1, mRNA under normal culture conditions. Cells were cultured under normoxic (N; 21%O2) or hypoxic (H; <2%O2) conditions for 1, 3, 6, 12 or 24 hours and total RNA was isolated, reverse transcribed and resultant cDNA was used to assess changes in PIGF mRNA expression by end-point PCR (figure). After optimization, the induction of PIGF mRNA expression at 12/24 hours of hypoxia were confirmed using real-time PCR: 12 hour = 3.88 ± 0.62 fold increase (n=6; p<0.05); 24 hour = 3.64 ± 0.41 fold increase (n = 6; p< 0.05). The results demonstrate very good concordance between real-time and end-point PCR. The main advantages of real-time over end-point PCR are: 1) its ability to measure DNA concentrations over a wider range, 2) higher throughput, 3) more quantitative than end-point PCR, and 4) no post-PCR handling is necessary. In conclusion, real-time PCR can accommodate for a wider variance in initial gene expression between samples and allows for more in-depth and rapid quantification of gene expression. en
dc.format.extent 3212076 bytes
dc.format.mimetype application/pdf
dc.language.iso en_US
dc.relation.ispartofseries DUCURS 2006;5
dc.subject Polymerase chain reaction en
dc.subject Gene expression en
dc.subject End-point PCR en
dc.subject Real-time PCR en
dc.subject PCR en
dc.title Assessing gene expression by real-time polymerase chain reaction en
dc.type Presentation en

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  • DUCURS [196]
    Poster sessions and presentation from the Drake University Conference on Undergraduate Research in the Sciences held each April at Olmsted Center on the Drake campus.

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