Assessing gene expression by real-time polymerase chain reaction

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.