Gene expression analysis by quantitative real-time PCR for floral tissues

Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental setup used. In addition, we provide protocols for performing qRT-PCR experiments in a multiwell plate format (with the LightCycler® 480 system, Roche) and with nanofluidic arrays (BioMark™ system, Fluidigm), which allow the automatic combination of sets of samples with sets of assays, and significantly reduce reaction volume and the number of liquid-handling steps performed during the experiment.Work in the authors’ laboratory is funded by grants from Spanish Ministerio de Economía y Competividad (BFU2011-22734; and Programa Consolider-Ingenio, CSD2007-00036), and the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (grant SGR2009-GRC476). J. J. was supported by a fellowship from the European Molecular Biology Organization (EMBO).Peer reviewe