Normalisation of genes of interest The use of nuclear- or plastid-encoded reference genes was evaluated for normalisation of two nuclear-encoded photosynthetic genes (ATPC and PSBO) and four plastid-encoded photosynthetic genes (PSAA, PSAB, PSBE and PETD). Remarkably, differences in gene expression levels were MAPK Inhibitor Library research buy observed depending on whether the data were normalised with nuclear- or plastid-encoded reference genes (Fig. 2).

For the transgenic 35S-CKX versus control tobacco plants, these differences were not as distinctive as for the Pssu-ipt versus control tobacco plants. In the latter, we clearly see that there is an influence of normalisation with nuclear- or plastid-encoded reference genes. These differences were also confirmed with the statistical Roxadustat solubility dmso analysis. For PSBE, PSAA, PSAB and PETD there is a significant difference (α = 0.05) between normalisation with plastid and nuclear normalisation factor. When normalizing the gene of interest with the plastid normalisation factor, we see that the gene expression is much lower (for Pssu-ipt) compared to normalisation with the nuclear normalisation factor (Fig. 2). Fig. 2 Gene expression levels normalized with nuclear (nuclear) or plastid (plastid) normalisation factor of selected genes of interest: PSBO (33 kDa subunit of the oxygen-evolving complex)

and ATPC (γ-subunit of ATP-synthase): nuclear encoded); PSBE (cytochrome b559), PSAA and PSAB (PSI-A and PSI-B) and PETD (subunit IV of cytochrome b 6 f) for Pssu-ipt (a) and 35S:CKX1 (b) expressed relatively Mirabegron to the wild-type control. Statistical significant differences (α = 0.05) are indicated (*) Discussion Real-time RT-PCR is an important technology to study changes in transcription levels. However, highly reliable reference genes are needed as internal controls for normalisation of the data. An internal control should show minimal changes, whereas

a gene of interest may change greatly during the course of an experiment (Dean et al. 2002). Choosing an internal control is one of the most critical steps in gene expression quantification. Vandesompele et al. (2002) showed that a conventional normalisation strategy, based on a single gene, led to erroneous normalisation. Using more internal reference genes, variation introduced by RNA sample quality, RNA input quantity and enzymatic efficiency in reverse transcription will be taken into account. In this study, we evaluated the expression stability of five nuclear-encoded and nine plastid-encoded reference genes in transgenic tobacco plants with elevated or diminished cytokinin content and their corresponding wild type. Analysis of the cytokinin content in these plants compared to the relative gene expression of the transgene clearly shows that overexpression of IPT or CKX has an effect on levels of the different cytokinin metabolites. This is in agreement with previous studies using Pssu-ipt or 35S:CKX1 transgenic tobacco plants (Synková et al.