Quantitative real time PCR in plant developmental biology.

Abstract

Gene expression patterns are important determinants of a cell's state, and changes in the expression profile indicate adaptation processes as a response to developmental transitions or environmental changes. Assaying gene expression can, therefore, help to elucidate mechanisms of determination and differentiation, as well as signaling networks. Several methods have been employed to determine transcript levels. The most quantitative and widely used technique is reverse transcription coupled to quantitative real time polymerase chain reaction (RT-qPCR). Live observation of fluorescence and, therefore, product increase during RT-qPCR allows the accurate determination of differences between initial template amounts. This is in contrast to the end-point analysis of conventional PCR, where initial differences in template amounts are usually masked because the analysis is done at the plateau phase. In the plateau phase, differences can no longer be distinguished due to inherent characteristics of PCR (e.g., loss of activity of the polymerase or because reaction components become limiting) that cause a drop in amplification efficiency, so that product accumulation levels out. Real time PCR circumvents this problem by shifting the analysis to an earlier stage of the amplification reaction.